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Week 27th June - 3rd July

Week 11th - 17th July

NEB Ph.D.™-7 Phage Display Peptide Library Kit solutions preparation

NEB protocol recommends to prepare a mixed solution of ITPG and X-gal. Because I already have IPTG stock, I prefer to do 2 separate solution for IPTG and X-gal (also avoids throwing both IPTG and X-gal if anything were to happen to the tubes).

IPTG

M.W.= 238.31g/mol
NEB protocol recommends to put 1.25g in 25mL DMF, which is [IPTG]=50mg/mL
[IPTG]mass=[IPTG]/MW=0.05/238.31=0.21mol/L=210mM.
They recommend diluting 1000-fold to prepare plates. So [IPTG]working_concentration=50µg/mL which is 210µM.
In order to simplify things, I'll use [IPTG]working_concentration=200µM.
IPTG stocks are at 1M and 0.1M (100mM).
If preparing 1M stock, add 2.383g of IPTG to 10mL of milli-Q water. For 0.1M, add 238.3mg of IPTG to 10mL of milli-Q water.
2µL IPTG / mL medium should be added if using the 0.1M solution (0.2µL / mL of using the 1M solution).

X-gal

M.W.= 408.63g/mol
NEB protocol recommends to put 1g in 25mL DMF, which is [X-gal]=40mg/mL.
Sigma-Aldrich recommends having stock solution at 20mg/mL.
I decided to do a stock at 20mg/mL.
2 µL X-gal / mL of medium should be added.

Tetracycline

NEB protocol recommends doing a 20mg/mL stock solution in 50% EtOH. Most of the protocol found online recommend 70% EtOH, so that's what I did (it probably does not matter much).
300mg were dissolved in 15mL 70% EtOH.

TBS

NEB protocol recommends 50mM Tris-HCl (pH7.5), 150mM NaCl.

Tris-HCl 1M, pH 7.5:
M.W. Tris-Base=121.14g/mol
12.11g of Tris-Base were dissolved in 60mL of H2O.
pH was adjusted to 7.5 using HCl 5M.
Volume was adjusted to 100mL.

25 mL of Tris-HCl 1M, pH 7.5 were diluted in 500mL H2O.
M.W. NaCl=58.44g/mol
mNaCl=(58.44/1000)*150=8.766g for 1L
So 8.766/2=4.383g were added to the solution of Tris-HCl 50mM.
pH was measured after completion of the solution and was around 7.7.

Blocking Buffer

M.W. NaHCO3=84.007g/mol
2.1g of NaHCO3 were put in 225mL osmosed H2O.
pH was adjusted to 8.6 with 5M HCl.
Volume was adjusted to 250mL.
1.25 BSA was added to final concentration 5mg/mL.
Solution was kept 6 days at 4°C.
On 21/07/16, 0.05g NaN3 (0.02%) were added to the solution to avoid growth of microorganisms.
Solution was filter sterilized and aliquoted by ~75mL.

Week 18th - 24th July

M13KE Phage Titering

In order to train ourselves with the phage titration that we will need to perform when doing the phage display from the NEB kit, we decided to do a phage titration using M13KE. The following protocol is the one given by NEB with their M13KE phages.

The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess).

Note: We used MGZ1 F+ to perform this titration

Inoculate 10 ml of LB with both MGZ1 F+ from a plate and incubate with shaking 4–6 hrs (mid-log phase, OD600 ~ 0.5).
Media was inoculated at 8h56.

Time after inoculation	OD 600nm
3h14	0.0584
4h11	0.2717
4h28	0.4022
4h28	0.5644

While cells are growing, melt Top Agar in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Maintain tubes at 45°C.
Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilution at 37°C until ready for use.
Prepare 10 to 1000-fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage culture supernatants, 10^8 –10^11 ; for unamplified panning eluates, 10^1–10^4. Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution.
As I expect to have 10^11 pfu/mL after amplification and that they recommend 10^8 to 10^11 dilutions from that, beginning from M13KE tube (10^13 pfu/mL), I used the following dilutions: 10^10, 10^11, 10^12 and 10^13 (shift of 2 order of magnitude).
When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution.
To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes. Here cells were incubated with the phages 3 to 4 minutes.
Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate. Gently tilt and rotate plate to spread top agar evenly.
Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.
Count plaques on plates that have approximately 100 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10 μl.

Results:

Dilution	Number of plaques
10^10	25
10^11	16
10^12	26
10^13	16

M13KE phage amplification

This phage amplification protocol is the one given by NEB.

Inoculate a 20 ml culture in a 250 ml Erlenmyer flask with 200 µL overnight E. coli culture. Add 1 µL phage suspension. Shake flask at 37°C, 150 rpm for 4 -5 hrs.
Remove cells by centrifugation at 4500 g for 10 min. Transfer supernatant to a fresh tube. Repeat centrifugation.
Transfer top 16 ml of supernatant to a new tube and add 4 mL of 2.5 M NaCl/20 % PEG-8000 (w/v). Briefly mix. Precipitate phage for 1 hr or overnight at 4°C.
Pellet phage by centrifugation at 12000 g for 15 min. 55 min at 4000 rpm (Rotor radius: 195 mm, 4000 rpm -> ~3400 g, 12000/~3400=3.5, 3.5*15 min=52.5 min) used was done instead due to problems with centrifuge. Decant supernatant. Resuspend pellet in 1 mL TBS. Transfer to an eppendorf tube. Spin briefly to remove any cell debris.
Transfer supernatant to a fresh tube. Add 200 µL of 2.5 M NaCl/20% PEG-8000. Incubate on ice for 60 min. Spin 14000 rpm in a benchtop centrifuge for 10 min. Discard supernatant. Spin again briefly and remove remaining supernatant with pipette. Resuspend pellet in 200 µL TBS.

Week 25th - 31th July

NEB Phage Display protocol on cotton - DAY 1

Cotton used: Khadi (from Khadi and Co - Bess Nielsen, given by Teja) - Hand Woven - 100% cotton.
A square of 1cm x 1cm was used (total surface: 2 cm² counting both sides).

Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 8 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
Incubate for at least 1 hour at 4°C a piece of fabric with blocking buffer.
Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.1% [v/v] Tween-20) by dipping the fabric in a beaker filled with TBST and going back and forth through the washing solution.
Dilute a 25-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
Rem: The square shape of the piece of fabric does not seem best suited for the incubaction with the phages as the sides seem not to be immersed in the phage library solution. It is probably better to use a rectangle shape thereafter so that the piece of fabric fits nicely into the tube and is fully immersed.
Remove piece of fabric from the Eppendorf tube.
Wash fabric 20 times with TBST as in step 3.
Elute bound phage with 1 ml of an appropriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
Inoculate 20 mL of LB medium in a 250-ml Erlenmeyer flask with ER2738. Incubate culture at 37°C with vigorous shaking. Carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD600 0.01–0.05), for use in Step 10.
Titer a small amount (~2 μl) of the eluate as described in General M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
Rem: As this eluate is not amplified, dilutions used for titration are as follow: 10, 100, 1000, 10000.
Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 1 (should be early-log at this point) and incubating with vigorous shaking for 4.5 hours at 37°C.
Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
Transfer the upper 80% (here 12.8mL) of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl (here ~2.1mL). Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.

Time after inoculation - Titration culture	OD 600nm - Titration culture	Time after inoculation - Amplification culture	OD 600nm - 20mL Amplification culture
0	0	0	0
3h30	0.3811	0h40	0.008/0.006/0.009
3h40	0.5101	1h50	0.0116
		2h25	0.0193

NEB Phage Display protocol on cotton - DAY 2

Results titration unamplified phage eluate 1st round:

Dilution	Number of plaques
10	>1000
100	230
1000	17
10000	3

Therefore, we have a phage concentration of 230x(10^2)x(10^2) = 2.3 x 10^6 pfu/mL.

Inoculate 10mL of LB+Tet medium with ER2738 for titration.
Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube.
Suspend the pellet in 1 ml of TBS. Transfer the suspension to a microcentrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl (here 166µL). Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pellet any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.

Titer the amplified eluate on LB/IPTG/Xgal plates following the general M13 titration protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C.
OD measurements:

Time after inoculation	OD 600nm
0	0
3h08	0.3167
3h21	0.4415
3h25	0.4955

Incubate overnight at 4°C a new piece of fabric (both a 1 cm x 1cm square and 0.5 cm x 2 cm were incubated) in 1 mL of Blocking Buffer for the 2 nd round of panning.

Week 01st - 07 August

NEB Phage Display protocol on cotton - DAY 3:

Count blue plaques from the titering plates in Step 17 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 4. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage.
Results titration amplified phage eluate 1st round:

Dilution	Number of plaques
10^8	>1000
10^9	92
10^10	8
10^11	1

Therefore, we have a phage concentration of 92x(10^9)x(10^2) = 9.2 x 10^12 pfu/mL.

Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 9 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.5% [v/v] Tween-20) by dipping the fabric in a beaker filled with TBST and going back and forth through the washing solution.
Dilute a 11.5-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
Rem: The square shape of the piece of fabric does not seem best suited for the incubaction with the phages as the sides seem not to be immersed in the phage library solution. It is probably better to use a rectangle shape thereafter so that the piece of fabric fits nicely into the tube and is fully immersed.
Remove piece of fabric from the Eppendorf tube.
Wash fabric 20 times with TBST as in step 3.
Elute bound phage with 1 ml of an appropriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
Inoculate 20 mL of LB medium in a 250-ml Erlenmeyer flask with ER2738. Incubate culture at 37°C with vigorous shaking. Carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD600 0.01–0.05), for use in Step 10.

Titer a small amount (~2 μl) of the eluate on LB/IPTG/Xgal following general M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
OD measuerments:

Time after inoculation	OD 600nm
0	0
3h33	0.1130
4h03	0.2107
4h35	0.3251
4h50	0.5356

Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 1 (should be early-log at this point) and incubating with vigorous shaking for 4.5 hours at 37°C.
OD measuerments:

Time after inoculation	OD 600nm
0	0
1h01	0
1h33	0.006
2h06	0.0388

Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
Transfer the upper 80% (here 14.4mL) of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl (here 2.4mL). Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.

NEB Phage Display protocol on cotton - DAY 4

Results titration unamplified phage eluate 2nd round:

Dilution	Number of plaques
10	>1000
100	788
1000	38
10000	10

Therefore, we have a phage concentration of 38x(10^3)x(10^2) = 3.8 x 10^6 pfu/mL

Inoculate 10mL of LB+Tet medium with ER2738 for titration.
Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube.
Suspend the pellet in 1 ml of TBS. Transfer the suspension to a microcentrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl (here 166µL). Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pellet any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.

Titer the amplified eluate on LB/IPTG/Xgal plates following the general M13 titration protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C.
OD measurements:

Time after inoculation	OD 600nm
0	0
3h38	1.032 => 10-fold dilution
4h18	0.2009
4h42	0.42
4h48	0.5094

NEB Phage Display protocol on cotton - DAY 5

Count blue plaques from the titering plates in Step 17 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 4. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage.
Results titration amplified phage eluate 1st round:

Dilution	Number of plaques
10^8	>1000
10^9	277
10^10	40
10^11	4

Therefore, we have a phage concentration of (277x(10^9)x(10^2)+40x(10^10)x(10^2))/2 = 3.36 x 10^13 pfu/mL

Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 8 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.5% [v/v] Tween-20) by dipping the fabric in a beaker filled with TBST and going back and forth through the washing solution.
Dilute a 168-fold representation of the library with 250µl of TBST (therefore add 1.5µL of phage library to 248.5µL TBST). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
Remove piece of fabric from the Eppendorf tube.
Wash fabric 20 times with TBST as in step 3.
Elute bound phage with 1 ml of an appropriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.

Titer a small amount (~2 μl) of the eluate on LB/IPTG/Xgal following general M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
OD measuerments:

Time after inoculation	OD 600nm
0	0
3h16	0.2936
3h45	0.2936
4h02	0.4242
4h11	0.5237

It is not necessary to amplify the third round eluate. Plaques from this titering can be used for sequencing: time the procedure so that plates are incubated at 37°C for no longer than 18 hours, as deletions may occur if plates are grown longer. Stored at 4°C, plates should be used to pick plaques within 1–3 days of plating. The remaining eluate can be stored at 4°C for at least one week.

Plaque amplification Sequencing - Cotton

Selected phage clones can be identified by DNA sequencing, and target specificity can be confirmed by phage ELISA. In both cases it is necessary to amplify phage, either from individual plaques or from the eluted pool, to obtain sufficient quantities to work with.

Dilute an overnight culture of ER2738 1:100 in LB. Dispense 1 ml of diluted culture into culture tubes, one for each clone to be characterized. 10–20 clones from the third round are usually sufficient to detect a consensus binding sequence. 20 clones were selected.
Use a sterile wooden stick or pipette tip (tip used here) to stab a blue plaque from a titering plate (important: plates should be <1–3 days old, stored at 4°C and have <100 plaques) and transfer to a tube containing the diluted culture. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence.
Incubate the tubes at 37°C with shaking for 4.5–5 hours (4h45').
Transfer the cultures to microcentrifuge tubes, and microfuge at 14,000 rpm for 30 seconds. Transfer the supernatant to a fresh tube and re-spin. Using a pipette, transfer the upper 80% of the supernatant to a fresh tube. This is the amplified phage stock and can be stored at 4°C for several weeks with little loss of titer. For long-term storage (up to several years), dilute 1:1 with sterile glycerol and store at –20°C.

Week 08th - 14th August

Sequencing of Phage DNA from Cotton Phage Display

After the first centrifugation in Step 4 in the phage amplification protocol, transfer 500 μl of the phage-containing supernatant to a fresh microfuge tube.
Add 200 μl of 20% PEG/2.5 M NaCl. Invert several times to mix, and let stand for 10–20 minutes at room temperature.
Re-spin briefly. Carefully pipet away and discard any remaining supernatant.
Spin in a microfuge at 14,000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the pellet with 0.5 ml of 70% ethanol (stored at –20°C), re-spin, discard the supernatant, and dry pellet (here it was dried 1h at RT with ~15µL EtOH remaining after discarding supernatant).
Suspend the pellet in 30 μl of H2O. The template can be suspended in TE if desired, this is recommended for long-term storage. In TE buffer, the phage DNA should be stable indefinitely at –20°C.

Quantitate product by using a Nanodrop.
Note: Make sure to use single strand Dna measurement

Sample	Concentration (ng/µL)	260/280 ration
R3P1	52	ND
R3P2	57	ND
R3P3	14	ND
R3P4	68	2.46
R3P5	29	2.53
R3P6	61	2.00
R3P7	97	2.01
R3P8	61	1.97
R3P9	61	2.55
R3P10	41	2.16
R3P11	80	2.04
R3P12	64	2.04
R3P13	64	2.15
R3P14	66	2.02
R3P15	65	1.96
R3P16	44.5	2.05
R3P17	45	2.10
R3P18	50	2.00
R3P19	43	2.00
R3P20	53	1.90

All samples were sent to sequencing by GATC Biotech.
Results are available below:

Sequences Phage R3P6 to R3P15

Sequences Phage R3P1 to R3P5 and R3P16 to R3P20

NEB Phage Display on Cotton - Finding a consensus sequence

Sequences were analysed using Geneious 9.1.5.
The random peptide library sequence was found between the linker (motif: 5'-ACC TCC ACC-3') and the end of the pIII leader sequence (motif: 5'-AGA GTG AGA-3'). We used only the -96 primer (see sequence below) to sequence as the -28 seemed too close to the library sequence for the sequencing to give a good result.

Some samples (R3P4, R3P13, R3P17) did not exhibit those motifs. Deletion?
One of the sequences was longer (22 nucleotides) and contained non-determined nucleotides (N).
Those four sequences were not used in the analysis.
The 21 nucleotides of the library of 16 remaining sequences were then translated.

Using the Multiple-Align function of Geneious and setting the threshold for the consensus sequence at 25% (amino acid present at least 25% of the time at a given position), we get the following consensus sequence:

Consensus_sequence_random_peptide_library_NEB_PhD_Cotton

NEB Phage Display on Polyester and Silk - DAY 1

Silk used: Silk thread 1003 Au ver a Soie - Colour: Crème (Cream) - 100% Soie - Length: 50 cm - Weight: 16.5mg.
Polyester used: Polyester thread Mediac Ref: 960 - Colour: 400 - 100% Polyester - Length: 50cm - Weight: 16mg.

DAY 1 from Panning Procedure (see protocol page) was followed. Amplification was not started this day.

OD600 measurements of titration culture were as follow:

Time after inoculation	OD 600nm
3h45	0.4984 => dilution 200-fold
6h10	0.1111
6h52	0.3191
7h02	0.4210
7h07	0.4567
7h12	0.4885

10mL LB+Tet were inoculated with a single ER2738 colony at 37°C with shaking. This culture will be diluted 100-fold the next day to amplify the unamplified panning eluate.

NEB Phage Display on Polyester and Silk - DAY 2 (Part 1)

Results titration unamplified phage eluate 1st round:

Dilution	Number of plaques
	Silk	Polyester
10	864	504
100	75	34
1000	16	3
10000	5	0

End of DAY 1 (steps 9-11) was performed.

Week 15th - 21st August

NEB Phage Display on Polyester and Silk - DAY 2 (Part 2)

NEB Phage Display on Polyester and Silk - DAY 3

NEB Phage Display on Polyester and Silk - DAY 4

NEB Phage Display protocol on Wool and Linen - DAY 1

Wool used: Pure wool gauze twill fabric 100%wool 3.2 oz/linear yard- From Dharma trading.
Linen used: 100% bleached linen, 3.8 oz/linear yard- From Dharma trading.
A rectange of 0.5cm x 2cm was used (total surface: 2 cm² counting both sides).
Followed Day1 of the phage display protocol but some things to note -
During step7, I removed the fabric from the elution buffer and then I centrifuged the solution to remove the lint particles from the wool fabric and then neutralized the supernatant.
Note- Wool fabric used is loosely woven, so while washing it is coming apart, I did not have the same problem with linen.

Time after inoculation - Titration culture	OD 600nm
0	>0
2h 15	0.25
2h 35	0.43
2h 48	0.48

For amplification, 1:100 dilution of the overnight culture of ER2738 was used.

NEB Phage Display on Polyester and Silk - DAY 5

NEB Phage Display protocol on Wool and Linen - DAY 2

Results of 1st round unamplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10	>1000	>1000
100	87	357
1000	10	46
10000	0	1

Therefore, we have following phage concentration -
Wool - 87x(10^2)x(10^2) = 8.7 x 10^5 pfu/mL.
Linen - 357x(10^2)x(10^2) = 3.57 x 10^6 pfu/mL.
Followed Day2 protocol exactly.
OD measurements:

Time after inoculation	OD 600nm
0	0
3h30	2.42
Diluted 1:20
4h00	0.443
4h07	0.5409

Plaque amplification for Sequencing - Polyester and Silk

NEB Phage Display protocol on Wool and Linen - DAY 3

Results of 1st round amplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10^8	>1000	>1000
10^9	267	277
10^10	41	22
10^11	2	4

Therefore, we have following phage concentration -
Wool - 267x(10^9)x(10^2) = 2.67 x 10^13 pfu/mL.
Linen - 277x(10^9)x(10^2) = 2.77 x 10^13 pfu/mL.
Round 2 panning:
To get a final phage number per reaction approximately 2*10^11, add 10µl of the amplified phage solution in 240µl TBST(0.5% Tween 20) to get a final volume of 250µl for both wool and linen
Followed Day3 protocol of Phage display protocol.
OD measurement of Culture for Titration

Time after inoculation	OD 600nm
0	0
2h20	0.303
2h32	0.521
Diluted 1:5	Because of Hood availability
3h32	0.363
3h45	0.4759

OD measurements of Culture for Amplification

Time after inoculation	OD 600nm
	Wool	Linen
1h00	>0.0129	>0.009
1h25	0.0179	0.0139

NEB Phage Display protocol on Wool and Linen - DAY 4

Results of 2nd round unamplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10	48	>1000
100	4	150
1000	1	18
10000	0	1

Therefore, we have following phage concentration -
Wool - 48x10x(10^2) = 4.8 x 10^4 pfu/mL.
Linen - 150x(10^2)x(10^2) = 1.5 x 10^6 pfu/mL.
Followed Day4 of the protocol exactly..
OD measurements:

Time after inoculation	OD 600nm
0	0
3h05	0.421
3h10	0.534

NEB Phage Display protocol on Wool and Linen - DAY 5

Results of 2nd round amplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10^8	>1000	>1000
10^9	394	269
10^10	50	35
10^11	4	3

Therefore, we have following phage concentration -
Wool - 394x(10^9)x(10^2) = 3.94 x 10^13 pfu/mL.
Linen - 269x(10^9)x(10^2) = 2.69 x 10^13 pfu/mL.
Round 3 panning:
To get a final phage number per reaction approximately 2*10^11, add-
Wool - 5.1µl of the amplified phage solution in 244.9µl TBST(0.5% Tween 20) to get a final volume of 250µl
Linen - 10µl of the amplified phage solution in 240µl TBST(0.5% Tween 20) to get a final volume of 250µl
Followed Day5 protocol of Phage display protocol.
OD measurements:

Time after inoculation	OD 600nm
0	0
3h00	0.364
3h15	0.522

NEB Phage Display protocol on Wool and Linen - DAY 6

Results of 3rd round unamplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10	499	>1000
100	38	547
1000	5	58
10000	0	6

Therefore, we have following phage concentration -
Wool - 38x100x(10^2) = 3.8 x 10^5 pfu/mL.
Linen - 58x(10^3)x(10^2) = 5.8 x 10^6 pfu/mL.

Week 22nd - 28th August

Sequencing of Phage DNA from Phage Display on Polyester and Silk

NEB Phage Display on Polyester and Silk - Finding a consensus sequence

Week 29th August - 4th September

Week 5th September - 11th September

Week 12th September - 18th September

Week 19th September - 25th September

Week 26th September - 02nd October

Week 03rd October - 09th October

Week 10th October - 16th October

Week 17th October - 23rd October

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

+33 1 44 41 25 22/25

igem2016parisbettencourt@gmail.com
2016.igem.org

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+			color : rgb(255,255,255);
+			text-decoration : none;
+			font-size : 18px;
+			font-family: 'Open Sans', sans-serif;
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+.input {
+width : 80%;
+display : inline-block;
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-<head>
-</head>
 <body>
+	<div id="topheader">
+	</div>
+	<div id="subheader">
+		<table id="ancre">
+			<tr>
+				<td><a href="#ancre">Notebook</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Protocols">Protocols</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Bibliography">Bibliography</a></td>
+			</tr>
+		</table>
+	</div>
+<div id=subheader2 >
+		<table>
+			<tr>
+                                <td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Assay">Assay</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Microbiology">Microbiology</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Enzyme">Enzyme search</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Indigo">Mission Indigo</a></td>
+				<td><a href="#ancre2">Binding Domains</a></td>
+				<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Anthocyanin">Anthocyanin</a></td>
+			</tr>
+		</table>
+	</div>
+	<div id="ancre2">
+		<div class="input">
-	<body>
+			<h1 class="red">Week 27th June - 3rd July</h1>
-		<div id="topheader">
-		</div>
-		<div id=subheader>
-			<table>
-				<tr>
-					<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook">Notebook</a></td>
-					<td><a href="#ancre">Protocols</a></td>
-					<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Bibliography">Bibliography</a></td>
-				</tr>
-			</table>
-		</div>
-<div id="ancre">
-    <h2 class="red">Phage Display Protocols</h2>
+			<h1 class="red">Week 11th - 17th July</h1>
-        <p>
+                <h3>NEB Ph.D.™-7 Phage Display Peptide Library Kit solutions preparation</h3>
-            All protocols displayed here are identical to the ones provided in the NEB Ph.D.™-7 Phage Display Peptide Libraries Instruction Manual, except for the Panning Procedure that was adapted to our needs.
-        </p>
+                    <p>
+                        NEB protocol recommends to prepare a mixed solution of ITPG and X-gal. Because I already have IPTG stock, I prefer to do 2 separate solution for IPTG and X-gal (also avoids throwing both IPTG and X-gal if anything were to happen to the tubes).
-        <h4>Phage Titering</h2>
+                    </p>
-            <p>
+                        <h5>IPTG</h5>
-                The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence.
+                            <p>
-            </p>
+                                M.W.= 238.31g/mol <br>
+                                NEB protocol recommends to put 1.25g in 25mL DMF, which is [IPTG]=50mg/mL <br>
-            <ol>
+                                [IPTG]mass=[IPTG]/MW=0.05/238.31=0.21mol/L=210mM. <br>
-                <li>
+                                They recommend diluting 1000-fold to prepare plates. So [IPTG]working_concentration=50µg/mL which is 210µM. <br>
-                    Inoculate 10mL of LB with ER2738 from a plate and incubate with shaking ~3-4 hours (mid-log phase, OD600 ~ 0.5).
+                                In order to simplify things, I'll use [IPTG]working_concentration=200µM. <br>
-                </li>
+                                IPTG stocks are at 1M and 0.1M (100mM). <br>
-                <li>
+                                If preparing 1M stock, add 2.383g of IPTG to 10mL of milli-Q water. For 0.1M, add 238.3mg of IPTG to 10mL of milli-Q water. <br>
-                    While cells are growing, melt Top Agar in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Maintain tubes at 45°C.
+µL IPTG / mL medium should be added if using the 0.1M solution (0.2µL / mL of using the 1M solution).
-                </li>
+                            </p>
-                <li>
-                    Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilu¬tion at 37°C until ready for use.
+                        <h5>X-gal</h5>
-                </li>
+                            <p>
-                <li>
+                                M.W.= 408.63g/mol <br>
-                    Prepare 10 to 103 -fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage culture supernatants, 10^8 – 10^11; for unamplified panning eluates, 10^1–10^4. Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution.
+                                NEB protocol recommends to put 1g in 25mL DMF, which is [X-gal]=40mg/mL. <br>
-                </li>
+                                Sigma-Aldrich recommends having stock solution at 20mg/mL. <br>
-                <li>
+                                I decided to do a stock at 20mg/mL. <br>
-                    When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution.
+µL X-gal / mL of medium should be added. <br>
-                </li>
+                            </p>
-                <li>
-                    To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes.
+                        <h5>Tetracycline</h5>
-                </li>
+                            <p>
-                <li>
+                                NEB protocol recommends doing a 20mg/mL stock solution in 50% EtOH. Most of the protocol found online recommend 70% EtOH, so that's what I did (it probably does not matter much). <br>
-                    Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate (200µM IPTG, 30µg/mL X-gal). Gently tilt and rotate plate to spread top agar evenly.
+mg were dissolved in 15mL 70% EtOH.
-                </li>
+                            </p>
-                <li>
-                    Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.
+                        <h5>TBS</h5>
-                </li>
+                            <p>
-                <li>
+                                NEB protocol recommends 50mM Tris-HCl (pH7.5), 150mM NaCl.
-                    Count plaques on plates that have approximately 100 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10µL. Multiply by 100 to get the pfu/mL.
+                            </p>
-                </li>
-            </ol>
+                            <p>
+                                Tris-HCl 1M, pH 7.5: <br>
-        <h4>Panning Procedure</h4>
+                                M.W. Tris-Base=121.14g/mol <br>
+.11g of Tris-Base were dissolved in 60mL of H2O. <br>
-            <p>
+                                pH was adjusted to 7.5 using HCl 5M. <br>
-                This protocol was adapted from the Surface Panning Procedure (Direct Target Coating) supplied in the NEB Ph.D.™-7 Phage Display Libraries Instruction Manual.
+                                Volume was adjusted to 100mL.
-            </p>
+                            </p>
-            <h5>DAY 0</h5>
+                            <p>
-                <ul>
+mL of Tris-HCl 1M, pH 7.5 were diluted in 500mL H2O. <br>
-                    <li>
+                                M.W. NaCl=58.44g/mol <br>
-                        Streak ER2738 from glycerol stock on LB+Tet plate.
+                                mNaCl=(58.44/1000)*150=8.766g for 1L <br>
-                    </li>
+                                So 8.766/2=4.383g were added to the solution of Tris-HCl 50mM. <br>
-                    <li>
+                                pH was measured after completion of the solution and was around 7.7.
-                        Incubate overnight at 4°C the piece of fabric/thread with 1mL of blocking buffer. Alternatively, after inoculation of the titering culture incubate the piece of fabric/thread at least 1 hour at 4°C on DAY 1.
+                            </p>
-                    </li>
-                 </ul>
+                        <h5>Blocking Buffer</h5>
+                            <p>
-            <h5>DAY 1</h5>
+                                M.W. NaHCO3=84.007g/mol <br>
+.1g of NaHCO3 were put in 225mL osmosed H2O. <br>
+                                pH was adjusted to 8.6 with 5M HCl. <br>
+                                Volume was adjusted to 250mL. <br>
+.25 BSA was added to final concentration 5mg/mL. <br>
+                                Solution was kept 6 days at 4°C. <br>
+                                On 21/07/16, 0.05g NaN3 (0.02%) were added to the solution to avoid growth of microorganisms. <br>
+                                Solution was filter sterilized and aliquoted by ~75mL. <br>
+                            </p>
+			<h1 class="red">Week 18th - 24th July</h1>
+                <h3>M13KE Phage Titering</h3>
+                    <p>
+                        In order to train ourselves with the phage titration that we will need to perform when doing the phage display from the NEB kit, we decided to do a phage titration using M13KE. The following protocol is the one given by NEB with their M13KE phages.
+                    </p>
+                    <p>
+                        The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess).
+                    </p>
+                    <p>
+                        Note: We used MGZ1 F+ to perform this titration
+                    </p>
+                    <ol>
+                        <li>
+                            Inoculate 10 ml of LB with both MGZ1 F+ from a plate and incubate with shaking 4–6 hrs (mid-log phase, OD600 ~ 0.5). <br>
+                            Media was inoculated at 8h56.
+                                <table style="width:90%" align="center">
+                                    <tr>
+                                        <th align="center">Time after inoculation</th>
+                                        <th align="center">OD 600nm</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h14</td>
+                                        <td align="center">0.0584</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h11</td>
+                                        <td align="center">0.2717</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h28</td>
+                                        <td align="center">0.4022</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h28</td>
+                                        <td align="center">0.5644</td>
+                                    </tr>
+                                </table>
+                        </li>
+                        <li>
+                            While cells are growing, melt Top Agar in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Maintain tubes at 45°C.
+                        </li>
+                        <li>
+                            Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilution at 37°C until ready for use.
+                        </li>
+                        <li>
+                            Prepare 10 to 1000-fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage culture supernatants, 10^8 –10^11 ; for unamplified panning eluates, 10^1–10^4. Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution. <br>
+                            As I expect to have 10^11 pfu/mL after amplification and that they recommend 10^8 to 10^11 dilutions from that, beginning from M13KE tube (10^13 pfu/mL), I used the following dilutions: 10^10, 10^11, 10^12 and 10^13 (shift of 2 order of magnitude).
+                        </li>
+                        <li>
+                            When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution.
+                        </li>
+                        <li>
+                            To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes. Here cells were incubated with the phages 3 to 4 minutes.
+                        </li>
+                        <li>
+                            Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate. Gently tilt and rotate plate to spread top agar evenly.
+                        </li>
+                        <li>
+                            Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.
+                        </li>
+                        <li>
+                            Count plaques on plates that have approximately 100 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10 μl.
+                        </li>
+                    </ol>
+                    <p>Results:</p>
+                        <table style="width:90%" align="center">
+                            <tr>
+                                <th align="center">Dilution</th>
+                                <th align="center">Number of plaques</th>
+                            </tr>
+                            <tr>
+                                <td align="center">10^10</td>
+                                <td align="center">25</td>
+                            </tr>
+                            <tr>
+                                <td align="center">10^11</td>
+                                <td align="center">16</td>
+                            </tr>
+                            <tr>
+                                <td align="center">10^12</td>
+                                <td align="center">26</td>
+                            </tr>
+                            <tr>
+                                <td align="center">10^13</td>
+                                <td align="center">16</td>
+                            </tr>
+                        </table>
+                 <h3>M13KE phage amplification</h3>
+                <p>
+                    This phage amplification protocol is the one given by NEB.
+                </p>
                  <ol>
                      <li>
-                         Inoculate 10 ml of LB+Tet medium with ER2738 in a 125mL Erlenmeyer flask. This culture will be used for titering in Step 8 and can be used in ~3.5 - 4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
+                         Inoculate a 20 ml culture in a 250 ml Erlenmyer flask with 200 µL overnight E. coli culture. Add 1 µL phage suspension. Shake flask at 37°C, 150 rpm for 4 -5 hrs.
-                    </li>
-                    <li>
-                        Remove the piece of fabric/thread from the blocking solution. Wash the fabric rapidly 10 times with TBST 0.1% (TBS + 0.1% [v/v] Tween-20) by dipping the threads in a well from a six-well plate filled with ~8mL TBST per well and going back and forth through the washing solution. Alternatively beackers can be used in place of six well plate.
-                    </li>
-                    <li>
-                        Dilute a 25-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Place the piece of fabric/thread in the Eppendorf tube containing the library and rock gently for 60 minutes at room temperature.
                      </li>
                      <li>
-                         Inoculate 20mL of LB medium in a 250mL Erlenmeyer flask just before the end of the incubation period of the fabric in the phage library solution. Incubate at 37°C with vigorous shaking. This culture will be used to amplify the eluted phages.
+                         Remove cells by centrifugation at 4500 g for 10 min. Transfer supernatant to a fresh tube. Repeat centrifugation.
                      </li>
                      <li>
-                         Remove piece of fabric/thread from the Eppendorf tube.
+                         Transfer top 16 ml of supernatant to a new tube and add 4 mL of 2.5 M NaCl/20 % PEG-8000 (w/v). Briefly mix. Precipitate phage for 1 hr or overnight at 4°C.
                      </li>
                      <li>
-                         Wash fabric 20 times with TBST as in step 2.
+                         Pellet phage by centrifugation at 12000 g for 15 min. 55 min at 4000 rpm (Rotor radius: 195 mm, 4000 rpm -> ~3400 g, 12000/~3400=3.5, 3.5*15 min=52.5 min) used was done instead due to problems with centrifuge. Decant supernatant. Resuspend pellet in 1 mL TBS. Transfer to an eppendorf tube. Spin briefly to remove any cell debris.
                      </li>
                      <li>
-                         Elute bound phage with 1 ml of an ap¬propriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric. Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
+                         Transfer supernatant to a fresh tube. Add 200 µL of 2.5 M NaCl/20% PEG-8000. Incubate on ice for 60 min. Spin 14000 rpm in a benchtop centrifuge for 10 min. Discard supernatant. Spin again briefly and remove remaining supernatant with pipette. Resuspend pellet in 200 µL TBS.
-                    </li>
-                    <li>
-                        Titer a small amount (~2 μl) of the eluate as described in the Phage Titering protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
-                    </li>
-                    <li>
-                        Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 4 (should be early-log at this point, i.e. OD600 ~ 0.01-0.05) and incubating with vigorous shaking for 4.5 hours at 37°C.
-                    </li>
-                    <em>
-                        Note: The remaining eluate can be stored overnight at 4°C at this point, if preferred, and amplified the next day. In this case, inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C. The next day, dilute the overnight culture 1:100 in 20 ml of LB in a 250-ml Erlenmeyer flask <strong>(do not use a 50 ml conical tube)</strong> and add the unamplified eluate. Incubate with vigorous shaking for 4.5 hours at 37°C and proceed to Step 10.
-                    </em>
-                    <li>
-                        Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
-                    </li>
-                    <li>
-                        Transfer the upper 80% of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl. Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.
                      </li>
                  </ol>
-            <h5>DAY 2</h5>
+			<h1 class="red">Week 25th - 31th July</h1>
-                <ol>
+                <h3>NEB Phage Display protocol on cotton - DAY 1</h3>
-                    <li>
-                        Inoculate 10mL of LB+Tet medium with ER2738 for titration.
+                    <p>
-                    </li>
+                        Cotton used: Khadi (from Khadi and Co - Bess Nielsen, given by Teja) - Hand Woven - 100% cotton. <br>
-                     <li>
+                        A square of 1cm x 1cm was used (total surface: 2 cm² counting both sides).
-                         Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube.
+                    </p>
-                    </li>
-                    <li>
+                    <ol>
-                        Suspend the pellet in 1 ml of TBS. Transfer the suspension to a micro¬centrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
+                        <li>
-                    </li>
+                            Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 8 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
-                    <li>
+                        </li>
-                        Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl. Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
+                        <li>
-                    </li>
+                            Incubate for at least 1 hour at 4°C a piece of fabric with blocking buffer.
-                    <li>
+                        </li>
-                        Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pel¬let any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.
+                        <li>
-                    </li>
+                            Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.1% [v/v] Tween-20) by dipping the fabric in a beaker filled with TBST and going back and forth through the washing solution.
-                    <li>
+                        </li>
-                        Titer the amplified eluate as described in the Phage Titering protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C.
+                        <li>
-                    </li>
+                            Dilute a 25-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature. <br>
-                    <li>
+                                Rem: The square shape of the piece of fabric does not seem best suited for the incubaction with the phages  as the sides seem not to be immersed in the phage library solution. It is probably better to use a rectangle shape thereafter so that the piece of fabric fits nicely into the tube and is fully immersed.
-                        Incubate overnight at 4°C a new piece of fabric/thread in 1mL of blocking buffer for the 2nd round of panning.
+                        </li>
-                    </li>
+                        <li>
-                    <li>
+                            Remove piece of fabric from the Eppendorf tube.
-                        Inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C.
+                        </li>
-                    </li>
+                        <li>
-                </ol>
+                            Wash fabric 20 times with TBST as in step 3.
+                        </li>
-            <h5>DAY 3</h5>
+                        <li>
+                            Elute bound phage with 1 ml of an appropriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
-                <ol>
+                        </li>
-                    <li>
+                        <li>
-                        Count blue plaques from the titering plates in Step 6 of DAY 2 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 3 of DAY 1. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage.
+                            Inoculate 20 mL of LB medium in a 250-ml Erlenmeyer flask with ER2738. Incubate culture at 37°C with vigorous shaking. Carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD600 0.01–0.05), for use in Step 10.
-                    </li>
+                        </li>
-                    <li>
+                        <li>
-                        Carry out a second round of panning: repeat DAY 1 using the calculated amount of the first round amplified eluate as input phage, and raising the Tween concentration in the wash steps to 0.5% (v/v).
+                            Titer a small amount (~2 μl) of the eluate as described in General M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired. <br>
-                    </li>
+                                Rem: As this eluate is not amplified, dilutions used for titration are as follow: 10, 100, 1000, 10000.
-                </ol>
+                        </li>
+                        <li>
-            <h5>DAY 4</h5>
+                            Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 1 (should be early-log at this point) and incubating with vigorous shaking for 4.5 hours at 37°C.
+                        </li>
-                <ol>
+                        <li>
-                    <li>
+                            Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
-                        Repeat DAY 2 with the panning eluate from the 2nd round.
+                        </li>
-                    </li>
+                        <li>
-                    <li>
+                            Transfer the upper 80% (here 12.8mL) of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl (here ~2.1mL). Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.
-                        Incubate overnight at 4°C a new piece of fabric/thread with 1mL of blocking buffer for the 3rd round of panning.
+                        </li>
-                    </li>
+                     </ol>
-                </ol>
+                <table style="width:90%" align="center">
-            <h5>DAY 5</h5>
+                    <tr>
+                        <th align="center">Time after inoculation - Titration culture</th>
-                <ol>
+                        <th align="center">OD 600nm - Titration culture</th>
-                    <li>
+                        <th align="center">Time after inoculation - Amplification culture</th>
-                        Carry out a third round of panning: repeat DAY 1 (steps 1 to 9 only), using the second round amplified eluate at an input titer equivalent to what was used in the first round, again using 0.5% Tween in the wash steps. It is not necessary to amplify the third round eluate. <br>
+                        <th align="center">OD 600nm - 20mL Amplification culture</th>
-                        <em>
+                    </tr>
-                            Plaques from the titering can be used for sequencing: time the procedure so that plates are incubated at 37°C for no longer than 18 hours, as deletions may occur if plates are grown longer. Stored at 4°C, plates should be used to pick plaques within 1–3 days of plating. The remaining eluate can be stored at 4°C for at least one week.
+                    <tr>
-                        </em>
+                        <td align="center">0</td>
-                    </li>
+                        <td align="center">0</td>
-                    <li>
+                        <td align="center">0</td>
-                        For preparation of individual clones for sequencing or ELISA, set up a 10-ml overnight culture of ER2738 from a colony, not by diluting the titering culture. Proceed with plaque amplification. Do not amplify the third round eluate and carry out a fourth round of panning unless the characterization shows no clear consensus sequence or phage ELISA results are negative.
+                        <td align="center">0</td>
-                    </li>
+                    </tr>
-                </ol>
+                    <tr>
+                        <td align="center">3h30</td>
-        <h4>Plaque Amplification for ELISA or Sequencing</h4>
+                        <td align="center">0.3811</td>
+                        <td align="center">0h40</td>
-            <p>
+                        <td align="center">0.008/0.006/0.009</td>
-                Selected phage clones can be identified by DNA sequencing, and target specificity can be confirmed by phage ELISA. In both cases it is necessary to amplify phage, either from individual plaques or from the eluted pool, to obtain sufficient quantities to work with.
+                    </tr>
-            </p>
+                    <tr>
+                        <td align="center">3h40</td>
+                        <td align="center">0.5101</td>
+                        <td align="center">1h50</td>
+                        <td align="center">0.0116</td>
+                    </tr>
+                    <tr>
+                        <td align="center"></td>
+                        <td align="center"></td>
+                        <td align="center">2h25</td>
+                        <td align="center">0.0193</td>
+                    </tr>
+                </table>
+                <h3>NEB Phage Display protocol on cotton - DAY 2</h3>
+                    <p>
+                        Results titration unamplified phage eluate 1st round:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                        <tr>
+                            <td align="center">100</td>
+                            <td align="center">230</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1000</td>
+                            <td align="center">17</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10000</td>
+                            <td align="center">3</td>
+                        </tr>
+                    </table>
+                    <p>
+                        Therefore, we have a phage concentration of 230x(10^2)x(10^2) = 2.3 x 10^6 pfu/mL.
+                    </p>
+                    <ol>
+                        <li>
+                            Inoculate 10mL of LB+Tet medium with ER2738 for titration.
+                         </li>
+                        <li>
+                            Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube.
+                        </li>
+                        <li>
+                            Suspend the pellet in 1 ml of TBS. Transfer the suspension to a microcentrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
+                        </li>
+                        <li>
+                            Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl (here 166µL). Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
+                        </li>
+                        <li>
+                            Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pellet any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.
+                        </li>
+                        <li>
+                            Titer the amplified eluate on LB/IPTG/Xgal plates following the general M13 titration protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C. <br>
+                            OD measurements:
+                                <table style="width:90%" align="center">
+                                    <tr>
+                                        <th align="center">Time after inoculation</th>
+                                        <th align="center">OD 600nm</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">0</td>
+                                        <td align="center">0</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h08</td>
+                                        <td align="center">0.3167</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h21</td>
+                                        <td align="center">0.4415</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h25</td>
+                                        <td align="center">0.4955</td>
+                                    </tr>
+                                </table>
+                        </li>
+                        <li>
+                            Incubate overnight at 4°C a new piece of fabric (both a 1 cm x 1cm square and 0.5 cm x 2 cm were incubated) in 1 mL of Blocking Buffer for the 2 nd round of panning.
+                        </li>
+			<h1 class="red">Week 01st - 07 August</h1>
-            <ol>
+                <h3>NEB Phage Display protocol on cotton - DAY 3:</h3>
-                 <li>
-                     Dilute an overnight culture of ER2738 1:100 in LB. Dispense 1 ml of diluted culture into culture tubes, one for each clone to be characterized. 10–20 clones from the third round are usually sufficient to detect a consensus binding sequence.
+                        <ol>
-                </li>
+                            <li>
-                <li>
+                                Count blue plaques from the titering plates in Step 17 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 4. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage. <br>
-                    Use a sterile wooden stick or pipette tip to stab a blue plaque from a titering plate (important: plates should be less than 1–3 days old, stored at 4°C and have less than 100 plaques) and transfer to a tube containing the diluted culture. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence.
+                                Results titration amplified phage eluate 1st round:
-                </li>
+                                    <table style="width:90%" align="center">
-                <li>
+                                        <tr>
-                    Incubate the tubes at 37°C with shaking for 4.5–5 hours (no longer).
+                                            <th align="center">Dilution</th>
-                </li>
+                                            <th align="center">Number of plaques</th>
-                <li>
+                                        </tr>
-                    Transfer the cultures to microcentrifuge tubes, and microfuge at 14,000 rpm for 30 seconds (see next section to purify sequencing template). Transfer the supernatant to a fresh tube and re-spin. Using a pipette, transfer the upper 80% of the supernatant to a fresh tube. This is the amplified phage stock and can be stored at 4°C for several weeks with little loss of titer. For longterm storage (up to several years), dilute 1:1 with sterile glycerol and store at –20°C.
+                                        <tr>
-                </li>
+                                            <td align="center">10^8</td>
-            </ol>
+                                            <td align="center">>1000</td>
+                                        </tr>
-        <h4>Sequencing of phage DNA</h4>
+                                        <tr>
+                                            <td align="center">10^9</td>
-            <p>
+                                            <td align="center">92</td>
-                This extremely rapid procedure produces template of sufficient purity for manual or automated dideoxy sequencing, without the use of phenol or chromatography.
+                                        </tr>
-            </p>
+                                        <tr>
+                                            <td align="center">10^10</td>
-            <ol>
+                                            <td align="center">8</td>
-                <li>
+                                        </tr>
-                    Carry out the plaque amplification procedure described in the Phage Amplification protocol. After the first centrifugation in Step 4, transfer 500 µl of the phage-containing supernatant to a fresh microfuge tube.
+                                        <tr>
-                </li>
+                                            <td align="center">10^11</td>
-                <li>
+                                            <td align="center">1</td>
-                    Add 200 µl of 20% PEG/2.5 M NaCl. Invert several times to mix, and let stand for 10–20 minutes at room temperature.
+                                        </tr>
-                </li>
+                                    </table>
-                <li>
+                                Therefore, we have a phage concentration of 92x(10^9)x(10^2) = 9.2 x 10^12 pfu/mL.
-                    Microfuge at 14,000 rpm for 10 minutes at 4°C and discard the supernatant. Phage pellet may not be visible.
+                            </li>
-                </li>
+                            <li>
-                <li>
+                                Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 9 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
-                    Re-spin briefly. Carefully pipet away and discard any remaining supernatant.
+                            </li>
-                </li>
+                            <li>
-                <li>
+                                Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.5% [v/v] Tween-20) by dipping the fabric in a beaker filled with TBST and going back and forth through the washing solution.
-                    Suspend the pellet thoroughly in 100 µl of Iodide Buffer by vigorously tapping the tube. Add 250 µl of ethanol and incubate 10–20 minutes at room temperature. Short incubation at room temperature will preferentially precipitate single-stranded phage DNA, leaving most phage protein in solution.
+                            </li>
-                </li>
+                            <li>
-                <li>
+                                Dilute a 11.5-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature. <br>
-                    Spin in a microfuge at 14,000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the pellet with 0.5 ml of 70% ethanol (stored at –20°C), re-spin, discard the supernatant, and briefly dry the pellet under vacuum.
+                                    Rem: The square shape of the piece of fabric does not seem best suited for the incubaction with the phages  as the sides seem not to be immersed in the phage library solution. It is probably better to use a rectangle shape thereafter so that the piece of fabric fits nicely into the tube and is fully immersed.
-                </li>
+                            </li>
-                <li>
+                            <li>
-                    Suspend the pellet in 30 µl of TE buffer. The template can be suspended in H2O instead of TE if desired, but this is not recommended for long-term storage. In TE buffer, the phage DNA should be stable indefinitely at –20°C.
+                                Remove piece of fabric from the Eppendorf tube.
-                </li>
+                            </li>
-                <li>
+                            <li>
-                    Quantitate product by using a Nanodrop.
+                                Wash fabric 20 times with TBST as in step 3.
-                </li>
+                            </li>
-            </ol>
+                            <li>
+                                Elute bound phage with 1 ml of an appropriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
-            <h5>Sequencing Guidelines</h5>
+                            </li>
+                            <li>
-                 <ol>
+                                Inoculate 20 mL of LB medium in a 250-ml Erlenmeyer flask with ER2738. Incubate culture at 37°C with vigorous shaking. Carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD600 0.01–0.05), for use in Step 10.
-                     <li>
+                            </li>
-                         The sequence being read corresponds to the anticodon strand of the template. Write out the complementary strand and check against the top strand of the insert sequence shown in the figure below (the primers hybridize downstream of the insert). Check that the third nucleotide of each codon in the randomized region is G or T.
+                            <li>
-                     </li>
+                                Titer a small amount (~2 μl) of the eluate on LB/IPTG/Xgal following general M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired. <br>
+                                OD measuerments:
+                                    <table style="width:90%" align="center">
+                                        <tr>
+                                            <th align="center">Time after inoculation</th>
+                                            <th align="center">OD 600nm</th>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">0</td>
+                                            <td align="center">0</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">3h33</td>
+                                            <td align="center">0.1130</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">4h03</td>
+                                            <td align="center">0.2107</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">4h35</td>
+                                            <td align="center">0.3251</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">4h50</td>
+                                            <td align="center">0.5356</td>
+                                        </tr>
+                                    </table>
+                            </li>
+                            <li>
+                                Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 1 (should be early-log at this point) and incubating with vigorous shaking for 4.5 hours at 37°C. <br>
+                                OD measuerments:
+                                    <table style="width:90%" align="center">
+                                        <tr>
+                                            <th align="center">Time after inoculation</th>
+                                            <th align="center">OD 600nm</th>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">0</td>
+                                            <td align="center">0</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">1h01</td>
+                                            <td align="center">0</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">1h33</td>
+                                            <td align="center">0.006</td>
+                                        </tr>
+                                        <tr>
+                                            <td align="center">2h06</td>
+                                            <td align="center">0.0388</td>
+                                        </tr>
+                                    </table>
+                            </li>
+                            <li>
+                                Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
+                            </li>
+                            <li>
+                                Transfer the upper 80% (here 14.4mL) of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl (here 2.4mL). Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.
+                            </li>
+                        </ol>
+                 <h3>NEB Phage Display protocol on cotton - DAY 4</h3>
+                    <p>
+                        Results titration unamplified phage eluate 2nd round:
+                    </p>
+                    <table style="width:90%">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                        <tr>
+                            <td align="center">100</td>
+                            <td align="center">788</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1000</td>
+                            <td align="center">38</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10000</td>
+                            <td align="center">10</td>
+                        </tr>
+                    </table>
+                    <p>
+                        Therefore, we have a phage concentration of 38x(10^3)x(10^2) = 3.8 x 10^6 pfu/mL
+                    </p>
+                    <ol>
+                        <li>
+                            Inoculate 10mL of LB+Tet medium with ER2738 for titration.
+                        </li>
+                        <li>
+                            Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube.
+                        </li>
+                        <li>
+                            Suspend the pellet in 1 ml of TBS. Transfer the suspension to a microcentrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
+                        </li>
+                        <li>
+                            Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl (here 166µL). Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
+                        </li>
+                        <li>
+                            Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pellet any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.
+                        </li>
+                        <li>
+                            Titer the amplified eluate on LB/IPTG/Xgal plates following the general M13 titration protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C. <br>
+                            OD measurements:
+                                <table style="width:90%" align="center">
+                                    <tr>
+                                        <th align="center">Time after inoculation</th>
+                                        <th align="center">OD 600nm</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">0</td>
+                                        <td align="center">0</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h38</td>
+                                        <td align="center">1.032 => 10-fold dilution</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h18</td>
+                                        <td align="center">0.2009</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h42</td>
+                                        <td align="center">0.42</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h48</td>
+                                        <td align="center">0.5094</td>
+                                    </tr>
+                                </table>
+                        </li>
+                     </ol>
+                <h3>NEB Phage Display protocol on cotton - DAY 5</h3>
+                    <ol>
+                        <li>
+                            Count blue plaques from the titering plates in Step 17 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 4. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage. <br>
+                            Results titration amplified phage eluate 1st round:
+                                <table style="width:90%" align="center">
+                                    <tr>
+                                        <th align="center">Dilution</th>
+                                        <th align="center">Number of plaques</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">10^8</td>
+                                        <td align="center">>1000</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">10^9</td>
+                                        <td align="center">277</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">10^10</td>
+                                        <td align="center">40</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">10^11</td>
+                                        <td align="center">4</td>
+                                    </tr>
+                                </table>
+                            Therefore, we have a phage concentration of (277x(10^9)x(10^2)+40x(10^10)x(10^2))/2 = 3.36 x 10^13 pfu/mL
+                        </li>
+                        <li>
+                            Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 8 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
+                        </li>
+                        <li>
+                            Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.5% [v/v] Tween-20) by dipping the fabric in a beaker filled with TBST and going back and forth through the washing solution.
+                        </li>
+                        <li>
+                            Dilute a 168-fold representation of the library with 250µl of TBST (therefore add 1.5µL of phage library to 248.5µL TBST). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
+                        </li>
+                        <li>
+                            Remove piece of fabric from the Eppendorf tube.
+                        </li>
+                        <li>
+                            Wash fabric 20 times with TBST as in step 3.
+                        </li>
+                        <li>
+                            Elute bound phage with 1 ml of an appropriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
+                        </li>
+                        <li>
+                            Titer a small amount (~2 μl) of the eluate on LB/IPTG/Xgal following general M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired. <br>
+                            OD measuerments:
+                                <table style="width:90%" align="center">
+                                    <tr>
+                                        <th align="center">Time after inoculation</th>
+                                        <th align="center">OD 600nm</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">0</td>
+                                        <td align="center">0</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h16</td>
+                                        <td align="center">0.2936</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h45</td>
+                                        <td align="center">0.2936</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h02</td>
+                                        <td align="center">0.4242</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">4h11</td>
+                                        <td align="center">0.5237</td>
+                                    </tr>
+                                </table>
+                        </li>
+                        <li>
+                            It is not necessary to amplify the third round eluate. Plaques from this titering can be used for sequencing: time the procedure so that plates are incubated at 37°C for no longer than 18 hours, as deletions may occur if plates are grown longer. Stored at 4°C, plates should be used to pick plaques within 1–3 days of plating. The remaining eluate can be stored at 4°C for at least one week.
+                        </li>
+                    </ol>
+                <h3>Plaque amplification Sequencing - Cotton</h3>
+                    <p>
+                        Selected phage clones can be identified by DNA sequencing, and target specificity can be confirmed by phage ELISA. In both cases it is necessary to amplify phage, either from individual plaques or from the eluted pool, to obtain sufficient quantities to work with.
+                    </p>
+                    <ol>
+                        <li>
+                            Dilute an overnight culture of ER2738 1:100 in LB. Dispense 1 ml of diluted culture into culture tubes, one for each clone to be characterized. 10–20 clones from the third round are usually sufficient to detect a consensus binding sequence. 20 clones were selected.
+                        </li>
+                        <li>
+                            Use a sterile wooden stick or pipette tip (tip used here) to stab a blue plaque from a titering plate (important: plates should be <1–3 days old, stored at 4°C and have <100 plaques) and transfer to a tube containing the diluted culture. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence.
+                        </li>
+                        <li>
+                            Incubate the tubes at 37°C with shaking for 4.5–5 hours (4h45').
+                        </li>
+                        <li>
+                            Transfer the cultures to microcentrifuge tubes, and microfuge at 14,000 rpm for 30 seconds. Transfer the supernatant to a fresh tube and re-spin. Using a pipette, transfer the upper 80% of the supernatant to a fresh tube. This is the amplified phage stock and can be stored at 4°C for several weeks with little loss of titer. For long-term storage (up to several years), dilute 1:1 with sterile glycerol and store at –20°C.
+                        </li>
+                    </ol>
+            <h1 class="red">Week 08th - 14th August</h1>
+                <h3>Sequencing of Phage DNA from Cotton Phage Display</h3>
+                    <ol>
+                        <li>
+                            After the first centrifugation in Step 4 in the phage amplification protocol, transfer 500 μl of the phage-containing supernatant to a fresh microfuge tube.
+                        </li>
+                        <li>
+                            Add 200 μl of 20% PEG/2.5 M NaCl. Invert several times to mix, and let stand for 10–20 minutes at room temperature.
+                        </li>
+                            Microfuge at 14,000 rpm for 10 minutes at 4°C and discard the supernatant. Phage pellet may not be visible.
+                        </li>
+                        <li>
+                            Re-spin briefly. Carefully pipet away and discard any remaining supernatant.
+                        </li>
+                            Suspend the pellet thoroughly in 100 μl of Iodide Buffer by vigorously tapping the tube. Add 250 μl of ethanol and incubate 10–20 minutes at room temperature. Short incubation at room temperature will preferentially precipitate single-stranded phage DNA, leaving most phage protein in solution.
+                        </li>
+                        <li>
+                            Spin in a microfuge at 14,000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the pellet with 0.5 ml of 70% ethanol (stored at –20°C), re-spin, discard the supernatant, and dry pellet (here it was dried 1h at RT with ~15µL EtOH remaining after discarding supernatant).
+                        </li>
+                        <li>
+                            Suspend the pellet in 30 μl of H2O. The template can be suspended in TE if desired, this is recommended for long-term storage. In TE buffer, the phage DNA should be stable indefinitely at –20°C.
+                        </li>
+                        <li>
+                            Quantitate product by using a Nanodrop. <br>
+                            Note: Make sure to use single strand Dna measurement
+                            <table style="width:90%">
+                                <tr>
+                                        <th align="center">Sample</th>
+                                        <th align="center">Concentration (ng/µL)</th>
+                                        <th align="center">260/280 ration</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P1</td>
+                                        <td align="center">52</td>
+                                        <td align="center">ND</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P2</td>
+                                        <td align="center">57</td>
+                                        <td align="center">ND</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P3</td>
+                                        <td align="center">14</td>
+                                        <td align="center">ND</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P4</td>
+                                        <td align="center">68</td>
+                                        <td align="center">2.46</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P5</td>
+                                        <td align="center">29</td>
+                                        <td align="center">2.53</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P6</td>
+                                        <td align="center">61</td>
+                                        <td align="center">2.00</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P7</td>
+                                        <td align="center">97</td>
+                                        <td align="center">2.01</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P8</td>
+                                        <td align="center">61</td>
+                                        <td align="center">1.97</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P9</td>
+                                        <td align="center">61</td>
+                                        <td align="center">2.55</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P10</td>
+                                        <td align="center">41</td>
+                                        <td align="center">2.16</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P11</td>
+                                        <td align="center">80</td>
+                                        <td align="center">2.04</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P12</td>
+                                        <td align="center">64</td>
+                                        <td align="center">2.04</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P13</td>
+                                        <td align="center">64</td>
+                                        <td align="center">2.15</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P14</td>
+                                        <td align="center">66</td>
+                                        <td align="center">2.02</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P15</td>
+                                        <td align="center">65</td>
+                                        <td align="center">1.96</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P16</td>
+                                        <td align="center">44.5</td>
+                                        <td align="center">2.05</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P17</td>
+                                        <td align="center">45</td>
+                                        <td align="center">2.10</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P18</td>
+                                        <td align="center">50</td>
+                                        <td align="center">2.00</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P19</td>
+                                        <td align="center">43</td>
+                                        <td align="center">2.00</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">R3P20</td>
+                                        <td align="center">53</td>
+                                        <td align="center">1.90</td>
+                                    </tr>
+                                </table>
+                        </li>
+                    </ol>
+                    <p>
+                        All samples were sent to sequencing by GATC Biotech. <br>
+                        Results are available below:
+                    </p>
+                    <a href="https://static.igem.org/mediawiki/2016/1/1f/Paris_Bettencourt-Sequencing_results_NEB_PhD_Cotton_6-15.zip">
+                        Sequences Phage R3P6 to R3P15
+                    </a> <br>
+                    <a href="https://static.igem.org/mediawiki/2016/e/ea/Paris_Bettencourt-Sequencing_results_NEB_PhD_Cotton_1-5_and_16-20.zip">
+                        Sequences Phage R3P1 to R3P5 and R3P16 to R3P20
+                    </a>
+                 <h3>NEB Phage Display on Cotton - Finding a consensus sequence</h3>
+                     <p>
+                        Sequences were analysed using Geneious 9.1.5. <br>
+                         The random peptide library sequence was found between the linker (motif: 5'-ACC TCC ACC-3') and the end of the pIII leader sequence (motif: 5'-AGA GTG AGA-3'). We used only the -96 primer (see sequence below) to sequence as the -28 seemed too close to the library sequence for the sequencing to give a good result.
+                     </p>
                      <div style="text-align:center;">
                          <img src="https://static.igem.org/mediawiki/2016/d/dd/Paris_Bettencourt-Sequencing_scheme.jpg" alt="Sequencing_scheme_NEB_PhD"/>
                      </div>
-                     <li>
-                         TAG stop codons are suppressed by glutamine in ER2738 (glnV). If the library was amplified in this strain or any glnV (also known as supE) strain, TAG should be considered a glutamine codon when translating.
+                     <p>
-                     </li>
+                         Some samples (R3P4, R3P13, R3P17) did not exhibit those motifs. Deletion? <br>
-                     <li>
+                        One of the sequences was longer (22 nucleotides) and contained non-determined nucleotides (N). <br>
-                         Libraries often contain a small percentage (less than 1%) of clones containing multiple inserts of the randomized region. Preferential selection and amplification of these clones may occur when panning against targets whose ligand specificity spans a length greater than that specified by the insert. When interpreting sequence data, be sure the sequence outside the restriction sites used for inserting the randomized sequence (Acc65I and EagI) matches the sequence shown in Figure 5. Once the flanking regions of the library insert have been identified, the insert size is easily determined.
+                        Those four sequences were not used in the analysis. <br>
-                     </li>
+                        The 21 nucleotides of the library of 16 remaining sequences were then translated.
-                 </ol>
+                     </p>
+                     <div style="text-align:center;">
+                         <img src="https://static.igem.org/mediawiki/2016/thumb/0/00/Paris_Bettencourt-Translation_sequence_random_peptide_library_NEB_PhD_Cotton.jpeg/800px-Paris_Bettencourt-Translation_sequence_random_peptide_library_NEB_PhD_Cotton.jpeg" alt="Translation_sequence_random_peptide_library_NEB_PhD_Cotton"/>
+                    </div>
+                    <p>
+                        Using the Multiple-Align function of Geneious and setting the threshold for the consensus sequence at 25% (amino acid present at least 25% of the time at a given position), we get the following consensus sequence:
+                    </p>
+                    <div style="text-align:center;">
+                        <img src="https://static.igem.org/mediawiki/2016/4/44/Paris_Bettencourt-Consensus_sequence_NEB_PhD_Cotton.jpg" alt="Consensus_sequence_random_peptide_library_NEB_PhD_Cotton"/>
+                     </div>
+                 <h3>NEB Phage Display on Polyester and Silk - DAY 1</h3>
+                    <p>
+                        Silk used: Silk thread 1003 Au ver a Soie - Colour: Crème (Cream) - 100% Soie - Length: 50 cm - Weight: 16.5mg.<br>
+                        Polyester used: Polyester thread Mediac Ref: 960 - Colour: 400 - 100% Polyester - Length: 50cm - Weight: 16mg.<br>
+                        <br>
+                        DAY 1 from Panning Procedure (see protocol page) was followed. Amplification was not started this day.<br>
+                        <br>
+                        OD600 measurements of titration culture were as follow:
+                    </p>
+                    <table style="width:90%" align="center">
+                                    <tr>
+                                        <th align="center">Time after inoculation</th>
+                                        <th align="center">OD 600nm</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">3h45</td>
+                                        <td align="center">0.4984 => dilution 200-fold</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">6h10</td>
+                                        <td align="center">0.1111</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">6h52</td>
+                                        <td align="center">0.3191</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">7h02</td>
+                                        <td align="center">0.4210</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">7h07</td>
+                                        <td align="center">0.4567</td>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">7h12</td>
+                                        <td align="center">0.4885</td>
+                                    </tr>
+                                </table>
+                    <p>
+mL LB+Tet were inoculated with a single ER2738 colony at 37°C with shaking. This culture will be diluted 100-fold the next day to amplify the unamplified panning eluate.
+                    </p>
-</div>
+                <h3>NEB Phage Display on Polyester and Silk - DAY 2 (Part 1)</h3>
+                    <p>
+                        Results titration unamplified phage eluate 1st round:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center" colspan="2">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Silk</th>
+                            <th align="center">Polyester</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10</td>
+                            <td align="center">864</td>
+                            <td align="center">504</td>
+                        </tr>
+                        <tr>
+                            <td align="center">100</td>
+                            <td align="center">75</td>
+                            <td align="center">34</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1000</td>
+                            <td align="center">16</td>
+                            <td align="center">3</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10000</td>
+                            <td align="center">5</td>
+                            <td align="center">0</td>
+                        </tr>
+                    </table>
+                    <p>
+                        End of DAY 1 (steps 9-11) was performed.
+                    </p>
+            <h1 class="red">Week 15th - 21st August</h1>
+                <h3>NEB Phage Display on Polyester and Silk - DAY 2 (Part 2)</h3>
+                <h3>NEB Phage Display on Polyester and Silk - DAY 3</h3>
+                <h3>NEB Phage Display on Polyester and Silk - DAY 4</h3>
+                <h3>NEB Phage Display protocol on Wool and Linen - DAY 1</h3>
+                    <p>
+                        Wool used: Pure wool gauze twill fabric 100%wool 3.2 oz/linear yard- From Dharma trading. <br>
+                        Linen used: 100% bleached linen, 3.8 oz/linear yard- From Dharma trading. <br>
+                        A rectange of 0.5cm x 2cm was used (total surface: 2 cm² counting both sides).<br>
+                        Followed Day1 of the phage display protocol but some things to note - <br>
+                        During step7, I removed the fabric from the elution buffer and then I centrifuged the solution to remove the lint particles from the wool fabric and then neutralized the supernatant.<br>
+                        Note- Wool fabric used is loosely woven, so while washing it is coming apart, I did not have the same problem with linen.
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Time after inoculation - Titration culture</th>
+                            <th align="center">OD 600nm</th>
+                        </tr>
+                        <tr>
+                            <td align="center">0</td>
+                            <td align="center">>0</td>
+                        </tr>
+                        <tr>
+                            <td align="center">2h 15</td>
+                            <td align="center">0.25</td>
+                        </tr>
+                        <tr>
+                            <td align="center">2h 35</td>
+                            <td align="center">0.43</td>
+                        </tr>
+                        <tr>
+                            <td align="center">2h 48</td>
+                            <td align="center">0.48</td>
+                        </tr>
+                    </table>
+                    <p>
+                        For amplification, 1:100 dilution of the overnight culture of ER2738 was used.
+                    </p>
+                <h3>NEB Phage Display on Polyester and Silk - DAY 5</h3>
+                <h3>NEB Phage Display protocol on Wool and Linen - DAY 2</h3>
+                    <p>
+                        Results of 1st round unamplified phage eluate titration:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center" colspan="2">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Wool</th>
+                            <th align="center">Linen</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10</td>
+                            <td align="center">>1000</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                        <tr>
+                            <td align="center">100</td>
+                            <td align="center">87</td>
+                            <td align="center">357</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1000</td>
+                            <td align="center">10</td>
+                            <td align="center">46</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10000</td>
+                            <td align="center">0</td>
+                            <td align="center">1</td>
+                        </tr>
+                    </table>
+                    <p>
+                        Therefore, we have following phage concentration - <br>
+                        Wool  -  87x(10^2)x(10^2) = 8.7 x 10^5 pfu/mL. <br>
+                        Linen - 357x(10^2)x(10^2) = 3.57 x 10^6 pfu/mL. <br>
+                        Followed Day2 protocol exactly.<br>
+                        OD measurements:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Time after inoculation</th>
+                            <th align="center">OD 600nm</th>
+                        </tr>
+                        <tr>
+                            <td align="center">0</td>
+                            <td align="center">0</td>
+                        </tr>
+                        <tr>
+                            <td align="center">3h30</td>
+                            <td align="center">2.42</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Diluted 1:20</td>
+                            <td align="center"> </td>
+                        </tr>
+                        <tr>
+                            <td align="center">4h00</td>
+                            <td align="center">0.443</td>
+                        </tr>
+                        <tr>
+                            <td align="center">4h07</td>
+                            <td align="center">0.5409</td>
+                        </tr>
+                    </table>
+                <h3>Plaque amplification for Sequencing - Polyester and Silk</h3>
+                <h3>NEB Phage Display protocol on Wool and Linen - DAY 3</h3>
+                    <p>
+                        Results of 1st round amplified phage eluate titration:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center" colspan="2">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Wool</th>
+                            <th align="center">Linen</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10^8</td>
+                            <td align="center">>1000</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                    <tr>
+                        <td align="center">10^9</td>
+                        <td align="center">267</td>
+                        <td align="center">277</td>
+                    </tr>
+                    <tr>
+                        <td align="center">10^10</td>
+                        <td align="center">41</td>
+                        <td align="center">22</td>
+                    </tr>
+                    <tr>
+                        <td align="center">10^11</td>
+                        <td align="center">2</td>
+                        <td align="center">4</td>
+                    </tr>
+                </table>
+                    <p>
+                        Therefore, we have following phage concentration - <br>
+                        Wool  - 267x(10^9)x(10^2) = 2.67 x 10^13 pfu/mL.<br>
+                        Linen - 277x(10^9)x(10^2) = 2.77 x 10^13 pfu/mL.<br>
+                        Round 2 panning:<br>
+                        To get a final phage number per reaction approximately 2*10^11, add 10µl of the amplified phage solution in              240µl TBST(0.5% Tween 20) to get a final volume of 250µl for both wool and linen <br>
+                        Followed Day3 protocol of Phage display protocol.<br>
+                        OD measurement of Culture for Titration
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                           <th align="center">Time after inoculation</th>
+                           <th align="center">OD 600nm</th>
+                        </tr>
+                        <tr>
+                            <td align="center">0</td>
+                            <td align="center">0</td>
+                        </tr>
+                        <tr>
+                            <td align="center">2h20</td>
+                            <td align="center">0.303</td>
+                         </tr>
+                         <tr>
+                            <td align="center">2h32</td>
+                            <td align="center">0.521</td>
+                         </tr>
+                         <tr>
+                            <td align="center">Diluted 1:5</td>
+                            <td align="center">Because of Hood availability</td>
+                         </tr>
+                         <tr>
+                            <td align="center">3h32</td>
+                            <td align="center">0.363</td>
+                         </tr>
+                         <tr>
+                            <td align="center">3h45</td>
+                            <td align="center">0.4759</td>
+                         </tr>
+                    </table>
+                    <p>
+                        OD measurements of Culture for Amplification
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Time after inoculation</th>
+                            <th align="center" colspan="2">OD 600nm</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Wool</th>
+                            <th align="center">Linen</th>
+                        </tr>
+                        <tr>
+                            <td align="center">1h00</td>
+                            <td align="center">>0.0129</td>
+                            <td align="center">>0.009</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1h25</td>
+                            <td align="center">0.0179</td>
+                            <td align="center">0.0139</td>
+                        </tr>
+                    </table>
+                <h3>NEB Phage Display protocol on Wool and Linen - DAY 4</h3>
+                    <p>
+                        Results of 2nd round unamplified phage eluate titration:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center" colspan="2">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Wool</th>
+                            <th align="center">Linen</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10</td>
+                            <td align="center">48</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                        <tr>
+                            <td align="center">100</td>
+                            <td align="center">4</td>
+                            <td align="center">150</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1000</td>
+                            <td align="center">1</td>
+                            <td align="center">18</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10000</td>
+                            <td align="center">0</td>
+                            <td align="center">1</td>
+                        </tr>
+                    </table>
+                    <p>
+                        Therefore, we have following phage concentration - <br>
+                        Wool  - 48x10x(10^2) = 4.8 x 10^4 pfu/mL.<br>
+                        Linen - 150x(10^2)x(10^2) = 1.5 x 10^6 pfu/mL.<br>
+                        Followed Day4 of the protocol exactly..<br>
+                        OD measurements:
+                    </p>
+                   <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Time after inoculation</th>
+                            <th align="center">OD 600nm</th>
+                        </tr>
+                        <tr>
+                            <td align="center">0</td>
+                            <td align="center">0</td>
+                        </tr>
+                        <tr>
+                            <td align="center">3h05</td>
+                            <td align="center">0.421</td>
+                        </tr>
+                        <tr>
+                            <td align="center">3h10</td>
+                            <td align="center">0.534</td>
+                         </tr>
+                    </table>
+                <h3>NEB Phage Display protocol on Wool and Linen - DAY 5</h3>
+                    <p>
+                        Results of 2nd round amplified phage eluate titration:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center" colspan="2">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Wool</th>
+                            <th align="center">Linen</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10^8</td>
+                            <td align="center">>1000</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10^9</td>
+                            <td align="center">394</td>
+                            <td align="center">269</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10^10</td>
+                            <td align="center">50</td>
+                            <td align="center">35</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10^11</td>
+                            <td align="center">4</td>
+                            <td align="center">3</td>
+                        </tr>
+                    </table>
+                    <p>
+                        Therefore, we have following phage concentration - <br>
+                        Wool  - 394x(10^9)x(10^2) = 3.94 x 10^13 pfu/mL.<br>
+                        Linen - 269x(10^9)x(10^2) = 2.69 x 10^13 pfu/mL.<br>
+                        Round 3 panning:<br>
+                        To get a final phage number per reaction approximately 2*10^11, add- <br>
+                        Wool  - 5.1µl of the amplified phage solution in 244.9µl TBST(0.5% Tween 20) to get a final volume of 250µl <br>
+                        Linen - 10µl of the amplified phage solution in 240µl TBST(0.5% Tween 20) to get a final volume of 250µl <br>
+                        Followed Day5 protocol of Phage display protocol.<br>
+                        OD measurements:
+                    </p>
+                   <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Time after inoculation</th>
+                            <th align="center">OD 600nm</th>
+                        </tr>
+                        <tr>
+                            <td align="center">0</td>
+                            <td align="center">0</td>
+                        </tr>
+                        <tr>
+                            <td align="center">3h00</td>
+                            <td align="center">0.364</td>
+                        </tr>
+                        <tr>
+                            <td align="center">3h15</td>
+                            <td align="center">0.522</td>
+                         </tr>
+                    </table>
+                <h3>NEB Phage Display protocol on Wool and Linen - DAY 6</h3>
+                    <p>
+                        Results of 3rd round unamplified phage eluate titration:
+                    </p>
+                    <table style="width:90%" align="center">
+                        <tr>
+                            <th align="center">Dilution</th>
+                            <th align="center" colspan="2">Number of plaques</th>
+                        </tr>
+                        <tr>
+                            <th align="center"> </th>
+                            <th align="center">Wool</th>
+                            <th align="center">Linen</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10</td>
+                            <td align="center">499</td>
+                            <td align="center">>1000</td>
+                        </tr>
+                        <tr>
+                            <td align="center">100</td>
+                            <td align="center">38</td>
+                            <td align="center">547</td>
+                        </tr>
+                        <tr>
+                            <td align="center">1000</td>
+                            <td align="center">5</td>
+                            <td align="center">58</td>
+                        </tr>
+                        <tr>
+                            <td align="center">10000</td>
+                            <td align="center">0</td>
+                            <td align="center">6</td>
+                        </tr>
+                    </table>
+                    <p>
+                        Therefore, we have following phage concentration - <br>
+                        Wool  - 38x100x(10^2) = 3.8 x 10^5 pfu/mL.<br>
+                        Linen - 58x(10^3)x(10^2) = 5.8 x 10^6 pfu/mL.<br>
+                    </p>
+            <h1 class="red">Week 22nd - 28th August</h1>
+                <h3>Sequencing of Phage DNA from Phage Display on Polyester and Silk</h3>
+                <h3>NEB Phage Display on Polyester and Silk - Finding a consensus sequence</h3>
+            <h1 class="red">Week 29th August - 4th September</h1>
+            <h1 class="red">Week 5th September - 11th September</h1>
+            <h1 class="red">Week 12th September - 18th September</h1>
+            <h1 class="red">Week 19th September - 25th September</h1>
+            <h1 class="red">Week 26th September - 02nd October</h1>
+            <h1 class="red">Week 03rd October - 09th October</h1>
+            <h1 class="red">Week 10th October - 16th October</h1>
+            <h1 class="red">Week 17th October - 23rd October</h1>
+		</div>
+	</div>
 </body>

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