Difference between revisions of "Team:Pasteur Paris/Microbiology week8"

Aim: Based on the results of silification tests, make a larger quantity of fusion protein.

Protocol: follow in this link

What we did in the lab:
Materials:

50 ml Falcon tube

Shaking incubator (INFORS HT)

Swing bucket centrifuge (JOUAN GR41)

Cultures of C1.2

carbenicillin at 50 mg/ml

LB medium

Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

; Ultrospec 3100

Method:

1.Put each of the C2 1.2 cultures in a 50 ml Falcon and centrifuge 8 minutes at 3500 RPM

2. Wash the pellet with 20 ml of LB after throwing away the supernatant

3. Centrifuge again 8 minutes at 3500 RPM and throw away the supernatant

4. Resuspend the pellet in 20 ml of LB

5. Pool the two Falcons

6. Put in the rotating incubator 4 Erlenmeyers of 2 L containing 1 L of LB, at 37°C and 150 RPM

7. Add in each Erlenmeyer 10 ml of C2 1.2 culture and 1 ml of carbenicillin at 50 mg/ml

8. Measure the OD_{600 nm} at several times :

Table 1
Times	OD_600nm
0	0.014
0 h 30	0.018
1 h 00	0.020
1 h 30	0.035
2 h 00	0.067
3 h 00	0.360
3 h 15	0.451
3 h 25	0.515
3 h 40	0.701

9. Pelleted the last measured sample for 3 minutes at 7000g. Then throw away the supernatant and store the pellet at &8722; 20 °C

10. Add 0.6 ml of iPTG in each Erlenmeyer and incubate overnight

Aim: DNA concentration measurement in order to find the correct ratios for ligation.

What we did in the lab:
Materials:
• Spectrophotometer
• Quartz cuvettes
• carbenicillin at 50 mg/ml
• LB medium
• B1 v2, B2 v2, C1 v2, C2 v2 DNA inserts
• Shaking incubator (INFORS HT)
• Microbiology equipment (37 °C incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
All the measurements are done twice to be more accurate
The preculture of vector is prepared with 3 ml of LB, 3 µl of carbenicillin and one colony of C2 1.2

Results

Table 2
DNA	pET 43.1 dephosphorylated		B1 v2		B2 v2		C1 v2		C2 v2
A₂₆₀	0.265	0.254	0.180	0.188	0.166	0.163	0.181	0.156	0.197	Ø
A₂₈₀	2.39	0.211	0.077	0.063	0.060	0.052	0.077	0.070	0.071	Ø
A_{260 ⁄ 280}	1.11	1.20	2.35	2.97	2.76	3.16	2.35	3.04	2.79	Ø
A_260⁄230	0.31	0.34	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
[ DNA] (ng ⁄ μl)	13.3	12.7	9	9.4	8.3	8.2	9	7.8	9.8	Ø

Aim: Prepare the ligation before doing the transformation with the Top10 competent cell .

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 1 ml Eppendorfs
• B1 v2 , B2 v2 , C1 v2 , C2 v2 DNA inserts
• Distilled water
• Shaking incubator (INFORS HT)
• T4 ligase
• T4 buffer

Method:
1. Prepare the following samples in a 1 ml Eppendorf, finishing with the ligase :

Table 3
	B1 v2 pET 43.1(a+) from 05 ⁄ 07	B2 v2 pET 43.1(a+) from 05 ⁄ 07	C1 v2 pET 43.1(a+) from 11 ⁄ 07	C2 v2 pET 43.1(a+) from 08 ⁄ 07
100 ng dephorphorylated ( μl)	6.7	6.7	7.7	7.7
Insert (16ng) (μl)	1.6	2	2	1.6
T4 ligase (μl)	1	1	1	1
T4 buffer (μl)	1.5	1.5	1.5	1.5
H₂O (μl)	4.2	3.8	1.8	3.2
V_final (μl)	15	15	15	15

2. Let ligate 1 hour at room temperature
3.Incubate 10 minutes at 65°C to inactivate the enzymes
4.Store all the samples at −80°C until ready to transform

Aim: Increase the amount of DNA.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipement
• Mastermix 2X
• Primer S (sense direction, Forward Primer, see sequence in PCR protocol section)
• Primers AS (anti-sense, Reverse Primer, see sequence in PCR protocol section)
• Phusion DNA polymerase
• DNA template
• Distilled water
• PCR machine

Method:
1. For each insert, make 3 tubes of 0.2 ml with thin walls and prepare the following mastermix :

Table 4
Volume (μl)	Insert	Primer S	Primer AS
Mastermix 2X	75	75	75
Primer S	3	3	Ø
Primer AS	3	Ø	3
H₂O	65.4	68.4	68.4
Phusion	0.6	0.6	0.6

2. Distribute the previous mastermix in the tubes and add the template DNA to have :

Table 5
Volume (μl)	Insert	Primer S	Primer AS	Without DNA
Mastermix	25	25	25	25
Primer S	1	1	Ø	1
Primer AS	1	Ø	1	1
Template DNA diluted 1⁄10	1	1	1	Ø
H₂O	21.8	22.8	22.8	22.8
Phusion	0.2	0.2	0.2	0.2
Total	50	50	50	50

3. Launch the PCR for 24 cycles

Aim: Get back plasmid DNA with the twinned insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• QIAGEN plasmid Midi Kit
• 2 ml Eppendorfs
• Spectrophotometer
• Ethanol 70%
• Cultures of BL21DE3 from the 25⁄07

Method:
1. When isopropanol is added, spread the volume in 5 x 2 (2 ml Eppendorfs) and centrifuge 10 minutes at 13000g
2. Add 1 ml of ethanol in each pellet and centrifuge 10 minutes at 13000g

Results:
Make 3 measurements of the concentration (1⁄200 dilution)

Table 6
	Measurement 1	Measurement 2	Measurement 3
OD₂₆₀	0.046	0.44	0.073
[DNA] (μg⁄ml)	460	4.40	730

Aim: Prepare our bacteria before the protein purification.

What we did in the lab:
Materials:
• Microbiological equipment
• PMSF at 100 mM
• Cultures of BL21DE3
• Buffer A
• 50 ml Falcons

Method:
1.Centrifuge 4x 1l of cultures 20 minutes at 4500g
2.Throw away the supernatant
3.Add 40 μl of PMSF in 40 ml of buffer A and add 5 ml of this solution in each pellet
4.Scratch the pellet to resuspend it
5.Add again 5 ml of the buffer A solution and mix the slurry until it becomes smooth, and to recover all the pellet
6.Put each product in a clean 50 ml Falcon : store at −80°C.

Table 7
Tubes	1	2	3	4
Weight (g)	15	15	15.3	16.1

Aim: Extract the bands C1 and C2 amplified with 900 bp inserts, and separate non-specific bands (700 bp).

Protocol: follow in this link

What we did in the lab:
Materials:
• QIAGEN Gel extraction Kit
• Buffer QG
• Gel bands C1 and C2
• Isopropanol
• 2 ml Eppendorfs

Method:
1. Calculate the amount of buffer QG for the extraction :

Table 8
	Weight of our tubes (mg)	Weight of our extraction (mg)	Buffer QG (μl)
C1	1.0360	40.2	121
C2	1.0154	32.8	97

2. Add 1 volume of isopropanol for C2 (32.3 μl) and 3 volumes of isopropanol for C1 (121 μl) 3. Recovery of DNA in 40 μl extraction buffer (20x2 μl)

Results:
Measurement of concentration with a dilution of 1⁄250 :

Table 9
	C1	C2
OD₂₆₀	0.003	0.003
OD₂₈₀	0.003	0.003
[DNA] (μg⁄ml)	30	120

Aim: To link vectors with the inserts.

Protocol: follow in this link

What we did in the lab:
Materials:
• Colonies C1 v2 and C2 v2 Insert bands
• Salt solution
• Distilled water
• TOPO vector

Method:

Table 10
	C1 v2	C2 v2
Colonies (μl)	4	4
Salt solution (μl)	1	1
Distilled water (μl)	5	5
TOPO vector (μl)	1	1
V_final (μl)	6	6

Aim: Save the bacteria with the recombinant vector for storage.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiological equipment
• Carbenicillin 50 μg⁄ml
• LB medium
• 50 ml Falcons
• 2 ml Eppendorfs
• Recombinant vector
• BL21DE3 colonies

Method:
1. Prepare in a falcon of 50 ml a preculture of the recombinant vector with one colony, 5 ml of LB and 5 μl of carbenicillin 50 mg⁄ml
2. Add 5 ml of LB and centrifuge 7 minutes at 3500 RPM
3. Discard the supernatant and add 5 ml of LB. Then, centrifuge again 7 minutes at 3500RPM
4. Discard the supernatant and add 5 ml of LB
5. Put 200 μl of this preparation into another Falcon of 50 ml, add 25 ml of LB and 25 μl of carbenicillin
6. Let the culture shake at 37°C for 4 hours (until the culture growth is sufficient, OD of approx. 0.7 at 600 nm) and check the concentration at several times during it :

Table 11
Time	OD_600nm
0	0.007
1h19	0.059
2h39	0.458
3h54	0.827

7. In 9 Eppendorfs of 2 ml, put 1 ml of LB with AGAR and 1 μl of carbenicillin. Then mix it
8. Dip a toothpick in the culture, and stab in each Eppendorf, and let it incubate at 37°C overnight. Afterwards store at 4°C.
9. On the July 28, 2016 : restreaking of cultures in a Petri dish (verification of STABs)

Aim: Separate the amplification bands of specific DNA (900pb) from non-specific bands (700pb).

Protocol: follow in this link

What we did in the lab:
Method:
We skipped this part to concentrate on the new versions of C1 and C2 inserts.

Aim: To determine if the two primers For (Forward direction) and Rev (Reverse direction) that were previously prepared are specific or not.

Protocol: follow in this link

What we did in the lab:
Materials:
• Mastermix 2X
• Primer S
• Primer AS
• Distilled water
• C1 and C2 inserts
• Phusion DNA polymerase
• Tubes for PCR
• PCR machine

Method:
1. For each inserts prepare the following Globalmix :

Table 12
Volume (μl)	Insert	Primer S	Primer AS
Mastermix 2X	75	75	75
Primer S	3	3	Ø
Primer AS	3	Ø	3
H₂O	65.4	68.4	68.4

2. Spread the previous samples and add DNA to have :

Table 13
Volume (μl)	Insert	Primer S	Primer AS	Without DNA
Globalmix	48.4	48.4	48.4	48.4
C1 or C2	1	1	1	1
Phusion	0.2	0.2	0.2	0.2
Total	50	50	50	50

3. Dispense these samples in 10 tubes and launch the PCR for 24 cycles

Aim: Check the results of the previous PCR of the new versions of C1 and C.

Protocol: follow in this link

What we did in the lab:
Materials:
• Ladder
• Inserts (old)
• C1 and C2
• Primer S
• Buffer EX
• Electrophoresis machine

Method:
Add 5 μl of DNA and 1 μl of lead buffer EX:

Table 14

Samples L1 L2 L3 L4 L5 L6 L7 L8

**Contain**
Ladder C1 + insert C1 + S C1 + AS C2 + insert C2 + S C2 + AS C without DNA

Results:
None of the bands of our samples can be seen because the temperature is not suitable (too hot). This experiment has to be redone another time at lower temperature.

Figure 1

Table 14
Samples	L1	L2	L3	L4	L5	L6	L7	L8
Contain	Ladder	C1 + insert	C1 + S	C1 + AS	C2 + insert	C2 + S	C2 + AS	C without DNA

Aim: PCR of the new versions of C1 and C2 at lower annealing temperature
Re-amplify some of the previous PCR reaction solution with fresh dNTPs, buffer and polymerase..

Protocol: follow in this link

What we did in the lab:
Materials:
• Samples from the previous PCR of the 26⁄07
• Tubes for PCR
• PCR machine
• dNTP
• MgCl₂ solution
• Phusion

Method:
1. Take 7 of the samples from the previous PCR (which contains only 45 μl after the electrophoresis)
2. With these samples, prepare the following samples by adding in order : dNTP, MgCl₂ and Phusion enzyme :

Table 15
Volume (μl)	Insert	Primer S	Primer AS	Without DNA
Samples	45	45	45	45
dNTP	6	6	6	6
MgCl₂	8.4	8.4	8.4	8.4
Phusion	0.5	0.5	0.5	0.5
V_final	60	60	60	60

3. Vortex each samples and centrifuge at room temperature at 3500 RPM
4. Run PCR following previous protocol, except for the lowered temperature.

Aim: Recovering the product of our previous PCR.

What we did in the lab:
Materials:
• QIAGEN Gel extraction Kit
• Products of the PCR of the 27/07
• 6X loading buffer
• Molecular weight DNA Ladder
• 10 μl pipette
• 200 μl pipette
• PCR tubes

Method:
1. Recovery from the agarose gel of 8 wells
2. Take 50 μl in PCR tubes and prepare several samples by adding in each tubes:
2.a 10 μl of 6X loading buffer and mix with one pipette
2.b 6 μl of ladder
3.Determination of the weight of the gel for extraction bands of C1 with For and Rev primers :
m(tube) = 1.1258g
m(tube+gel) = 1.2919
→ m(gel) = 165mg which correspond to 496.5 μl of buffer
4. Follow the QIAGEN gel extraction Kit protocol
Results :

Figure 2 1: Ladder
2: Without DNA
3: C1 insert Fer and Rev
4: C1 Fer
5: C1 Rev
6: C2 insert
7: C2 Fer
8: C2 Rev

Aim: Check if our new plasmid (vector TOPO + C1v2 and C2 v2 can be inserted in a bacteria.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiological equipment
• Carbenicillin at 50 mg⁄ml
• LB medium
• Petri dish
• TOPO10
• Dimethylformamide (DMF)
• Xgal 20 mg/ml in DMF

Method:
1. Prepare the solutionof Xgal for the Petri dish with :
1.a 10 ml of dimethylformamide
1.b 200 mg of Xgal
2. Put 100 μl of this solution on 6 Petri dish which already contain LB and carbenicillin. Then spread the solution in an additional volume of LB. Let the Petri dishes dry at 37°C for 30 minutes-1 hr. 3. Recovery C2 v2 and C1 v2 prepared the 26⁄07 (to perform the TOPO10 cloning reaction)
4. Put 50 μl of bacteria TOPO10 in ice and add 6 μl of C1 v2 or C2 v1
5. Follow the protocol to transform thermally competent bacteria

Aim: Know the concentration of the inserts.

What we did in the lab:
Materials:
See protocol

Result

Table 16
	C1 v2	C2 v2
V_TE (μl)	498	498
V_DNA (μl)	2	2
Ratio	X	X
A₂₆₀	0.002	0.001
A₂₈₀	0.001	0.001
[Insert] (μg⁄ml)	20	25

Aim: Send of the recombinant vector C2.1 (from the Midiprep of the 26⁄07) in sequencing to Eurofins-MWG.

Results:

Table 17
[DNA_C2.1] (ng⁄ml)	450
V_DNA (μl)	3.3
V_H2O (μl)	11.7
V_{primer d1⁄50} (μl)	2
V_total (μl)	17

Aim: Check if the transformation worked.

What we did in the lab :
Materials :
• Microbiological equipment
• 50 ml Erlenmeyer
• Incubator
• LB medium
• Carbenicillin at 50 mg⁄ml

Method
1. Only one colony had grown so we put it into a 50 ml Erlenmeyer with 25 ml of LB and 25 μl of carbenicillin
2. Put the Erlenmeyer into the incubator at 37°C with agitation overnight

Aim: Have Petri dishes ready for future transformations with TOPO10 cloning.

What we did in the lab :
Materials :
• Solution from TOPO10 transformation with the ligation TOPO cloning product (experiment from the 27⁄07)
• Dimethylformamide
• Xgal at 10 mg⁄ml
• 200 µl micropipette
• 10 ml pipette
• Petri dishes

Method
1. In a Petri dish, put 100 μl of the solution from TOPO10 transformation with the ligation TOPO cloning product
2. Add 10 ml of dimethylformamide and 10 mg of Xgal
3. Spread 200 μl of Xgal on the Petri dishes, and allow drying at 37°C for 30 minutes to 1 hour.
4. Carry out the transformation according to the thermal protocol

Aim: Know the concentration of purified C1 (For + Rev).

What we did in the lab :
Materials :
• Ulltrospec 3100
• Quartz cuvette
• TE buffer
• C1 DNA from the PCR purification (27⁄07)
• Petri dishes

Method
1. Do the blank with 500 μl of TE buffer
2. For the sample, use 498 μl of TE buffer and 2 μl of C1 DNA from the PCR purification

Results

Table 18
OD₂₆₀	OD₂₈₀	[DNA_C1] (μg⁄ml)
0.002	0.001	50

Aim: Protein purification.

Protocol: follow in this link

What we did in the lab :
Materials :
• FPLC machine (Akta Prime Plus, GE Lifesciences)
• 5 ml Ni-NTA His-trap column
• Buffer A (50 mM Tris pH 7.4 ; 150 mM NaCl), Buffer B=Buffer A + 250 mM Imidazole
• PMSF at 100 mM
• Branson 450 (sonicator)
• JA 25.5 rotor for Beckman Avanti X26-J centrifuge

Method
On ice :
1. Defrost one aliquot of C1 bacteria culture under cold running water
2. Equilibrate the column on FPLC machine with buffer A
3. Once the bacteria thawed out, add 22 ml of buffer A and 37μl of PMSF
4. Lyse the bacteria with a sonicator
5. Use a medium size tip, put the duty cycle at 60% and power under the limit of the micro tip setting
6. With the sonicator machine, do 3 times of 30 seconds of sonication follow-up with 1 minutes of rest on ice
7. Balance polycarbonate 50 ml tubes containing the lysate on a microbalance
8. Centrifuge the lysate at 16000 RPM in a JA 25.5 rotor in a Beckman Avanti X 26J at 4°C during 30 minutes
9. Transfer the supernatant in a 50 ml Falcon and add 37μl of PMSF
10. Inject 10 ml of this sample with a syringe equipped with a 0.45 μm filter in the injection 11; port and load it on the column (Ni-NTA His Trap, 5 ml, GE Lifesciences)
12. Wash the injection loop and column with buffer A until the UV detector output at 280 nm is close to the baseline and repeat the injection 3 times
13. Wash the column with 5% buffer B
14. Run a gradient from 5&#37 to 100&#37 buffer B over 12 column volumes and collect fractions of 1 ml
15. Wash the column with buffer A

“”/ — Aim: Revelation of the protein in each fraction/peak.

What we did in the lab :
Materials :
• 10X TGS buffer (Biorad)
• Distilled water
• Page Ruler Protein Molecular weight ladder (Thermofisher)
• 1 SDS PAGE gel, BioRad (TGS: Tris Glycine NaCl)
• Electrophoresis chamber , Power supply and electrodes (Biorad)
• 2X SDS (Laemli buffer with Beta-mercapto ethanol)
• 95°C Heater block
• Gel Code blue stain (Pierce) with G250 Coomassie

Method
1. At the beginning, prepare 1X from 10X TGS buffer with 25 ml of TGS buffer and 225 ml of distilled water
2. Heat samples at 95°C for 5 minutes, cool at RT, and load 20 μl in each well; 6 μl of ladder.
3. Run at 130 V

Results :
1 : Page ruler Ladder
2 : Well 19
3 : Well 21
4 : Welle 25
5 : Well 30
6 : Well 34
7 : Well 36
8 : Well 37
9 : Well 38
10 : Well 39
The concentration of proteins is too high so this experiment has to be redone

Figure 3

“”/ — Aim: Revelation of proteins in each well.

What we did in the lab :
Materials :
• TGS 10X
• PAGE ruler molecular weight marker (Thermofisher)
• Distilled water
• Protein fraction solutions from FPLC
• Buffer A
• Laemli 2X
• SDS PAGE gel, electrophoresis chamber, 3000 V power supply (Biorad)

Method
1. Prepare 400 ml of TGS 1X with 40 ml of TGS 10X and 360 ml of distilled water
2. Dilute protein solutions :

Table 19

Fractions from FPLC Dilution Contain

19, 21, 25 Ø Ø

30, 34, 36 (1⁄100) in buffer A 1μl of protein with 99μl of buffer A

37, 38, 39 (1⁄10) in buffer A 1μl of protein with 9μl of buffer A

3. In each gel well, add 10 μl of diluted protein solution and 10 μl of laemli 2X
4. Put all samples at 95°C during 5 minutes and follow the protocol for SDS PAGE gel

Results :
The distribution of samples in lanes is the same as the previous experiment:
Figure 4 1 : Ladder
2 : Well 19
3 : Well 21
4 : Well 25
5 : Well 30
6 : Well 34
7 : Well 36
8 : Well 37
9 : Well 38
10 :Well 39

Table 19
Fractions from FPLC	Dilution	Contain
19, 21, 25	Ø	Ø
30, 34, 36	(1⁄100) in buffer A	1μl of protein with 99μl of buffer A
37, 38, 39	(1⁄10) in buffer A	1μl of protein with 9μl of buffer A

Aim: Test the specificity of our two primers : For and Rev.

Protocol: follow in this link

What we did in the lab :
Materials :
• dNTP
• Solution of MgCl₂
• RNA free
• Buffer 10X pfu DNA polymerase
• Inserts C1 and C2
• For primer
• Rev primer
• 2 ml Eppendorfs
• Distilled water

Method
1. Make a global mix for the 7 samples :

Table 20
	Volumes (μl)
dNTP	22.5
MgCl₂	22.5
RNA free	327.6
Buffer 10X pfu	22.5
Total	417.6

2. Distribute the global mix in each tubes and add DNA :

Table 21
Tubes	Name	V(premix) (μl)	V(C1) (μl)	V(C2) (μl)	V(primer For) (μl)	V(primer Rev) (μl)	V(H₂O) (μl)
1	C1 insert For and Rev	46.4	1	0	1	1	0
2	C1 insert For	46.4	1	0	1	0	1
3	C1 insert Rev	46.4	1	0	0	1	1
4	C2 insert For and Rev	46.4	0	1	1	1	0
1	C2 insert For	46.4	0	1	1	0	1
1	C1 insert Rev	46.4	0	1	0	1	1
1	Without DNA	0	0	0	0	0	0

3. Put the tubes in a PCR machine heat to 94°C during 1 minute and then add the enzyme.

Aim: Test the specificity of our two primers : For and Rev.

Protocol: follow in this link

What we did in the lab :
Materials :
• 100 ml Erlenmeyer
• TAE 1X
• Agarose gel
• Ethidium bromide drops (EB)
• Gel electrophoresis apparatus
• Gene ruler ladder
• Loading buffer 6X
• DNA (C1 and C2, For and Rev)

Method
1. Prepare a 0.71% agarose gel :
1.a In an Erlenmeyer of 100 ml, put 50 ml of TAE1 X and add 0.35g of agarose
1.b Warm it in a microwave 2 minutes and let it cool down a little
1.c Add 2 drops of EB and mix it
1.d Transfer it on combs in a gel caster and let it solidify
2. Put samples in the wells:

Table 22

Wells Samples

1
7 μl of gene ruler

2
2 μl of 6X + 10 μl of DNA (C1 For and Rev)

3
2 μl of load 6X + 10 μl of DNA (C1 For)

4
2 μl of load 6X + 10 μl of DNA (C1 Rev)

5
2 μl of load 6X + 10 μl of DNA (C2 For and Rev)

6
2 μl of load 6X + 10 μl of DNA (C2 For)

7
2 μl of load 6X + 10 μl of DNA (C2 Rev)

8
2 μl of load 6X + 10 μl of distilled water

Results :

Figure 5 1 : Gene ruler 7 μl
2 : C1 For + Rev
3 : C1 For
4 : C1 Rev
5 : C2 For + Rev
6 : C2 For
7 : C2 Rev
8 : Without DNA
The experiments did not work.

Table 22
Wells	Samples
1	7 μl of gene ruler
2	2 μl of 6X + 10 μl of DNA (C1 For and Rev)
3	2 μl of load 6X + 10 μl of DNA (C1 For)
4	2 μl of load 6X + 10 μl of DNA (C1 Rev)
5	2 μl of load 6X + 10 μl of DNA (C2 For and Rev)
6	2 μl of load 6X + 10 μl of DNA (C2 For)
7	2 μl of load 6X + 10 μl of DNA (C2 Rev)
8	2 μl of load 6X + 10 μl of distilled water

Aim: Extracting DNA from the colony obtained after transformation.
Protocol: follow in this link

Materials :
• Miniprep QIAGEN Kit
• TOPO cloning colony
• Buffer EB
• Spectrophotometer

Method
1. For the Miniprep prepare 6x 2 ml of cultures and recover it in 50 μl of buffer EB
2. Do the prep with the QIAGEN Miniprep Kit
3. Measure the absorbance of the products to determine the amount of DNA obtained after extraction

Results :

Table 23
Samples	A₂₆₀	A₂₈₀	[DNA] (μg⁄ml)
1	0.009	0.001	120 → wrong
2	0.001	0.003	10
3	0.001	0.003	11

Aim:Check if the insert is in the TOPO vector.
Protocol: follow in this link

Materials :
• Biochemical equipment
• TOPO vector
• Xba I (NEB)
• Hind III (NEB)
• Buffer Cutsmart 10X (NEB)
• Distilled water

Method

Table 24
	Volumes (μl)
DNA (10 μg⁄ml)	5
XbaI	1
HindIII	1
Buffer Cutsmart 10X	4
H₂O	29
Total	40

Aim:Protein conservation.
Materials :
• Biochemical equipment
• Glycerol 80 %

Method
To prepare glycerol 80% with a final volume of 40 ml, use 32 ml of glycerol with 8 ml of H2O (40×80)⁄100 = 32ml

@@ Line 343: / Line 343: @@
                         </tbody>
                     </table><br/>
-. Pelleted the last measured sample for 3 minutes at 7000g. Then throw away the supernatant and store the pellet at &8722; 20 &#176;C <br/>
+<li>9. Pelleted the last measured sample for 3 minutes at 7000g. Then throw away the supernatant and store the pellet at &8722; 20 &#176;C <br/></li>
-. Add 0.6 ml of iPTG in each Erlenmeyer and incubate overnight<br/><br/><br/>
+<li>10. Add 0.6 ml of iPTG in each Erlenmeyer and incubate overnight<br/><br/><br/></li>
                 </p>
              </figcaption>
@@ Line 357: / Line 357: @@
                 <figcaption>
                    <p>
-      <U> Aim:</U> Have a measure of the DNA concentration in order to find the correct ratios for ligation.<br/> <br/>
+      <U> Aim:</U> DNA concentration measurement in order to find the correct ratios for ligation.<br/> <br/>
                    <U>What we did in the lab:</U><br/>
                    <U>Materials:</U><br/>
-                     &bull; Spectrophotometer <br/>
+                &bull; Spectrophotometer <br/>
 	        &bull; Quartz cuvettes <br/>
-	        &bull; Carbenicillin at 50 mg/ml <br/>
+	        &bull; carbenicillin at 50 mg/ml <br/>
-                     &bull; LB medium <br/>
+                &bull; LB medium <br/>
-              &bull; B1 v2, B2 v2, C1 v2, C2 v2 DNA inserts<br/>
+                &bull; B1 v2, B2 v2, C1 v2, C2 v2 DNA inserts<br/>
-                     &bull; Shaking incubator (INFORS HT)<br/>
+                &bull; Shaking incubator (INFORS HT)<br/>
-                     &bull; Microbiology equipment (37 &#176;C incubator, Bunsen burner, water bath, etc… Follow this link)<br/><br/>
+                &bull; Microbiology equipment (37 &#176;C incubator, Bunsen burner, water bath, etc… Follow this link)<br/><br/>
                    <U>Method:</U><br/>
        		All the measurements are done twice to be more accurate<br/>
-The preculture of vector is prepared with 3 ml of LB, 3 &#0181;l of carbenicillin and one colony of C2 1. <br/><br/>
+The preculture of vector is prepared with 3 ml of LB, 3 &#0181;l of carbenicillin and one colony of C2 1.2 <br/><br/>
 	       <U>Results</U><br/>
                    <table>
@@ Line 464: / Line 464: @@
            <a href="#" class="closemsg"></a>
               <figcaption>
-                  <p><U> Aim:</U> Prepare the ligation before doing the transformation with the competent cells Topo10.<br/><br/>
+                  <p><U> Aim:</U> Prepare the ligation before doing the transformation with the Top10 competent  cell .<br/><br/>
                       <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br/><br/>
                       <U>What we did in the lab:</U><br/>
                       <U>Materials:</U><br/>
-                          &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link) <br/>
+                   &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link) <br/>
                     &bull; 1 ml Eppendorfs <br/>
                     &bull; B1 v2 , B2 v2 , C1 v2 , C2 v2 DNA inserts <br/>
                     &bull; Distilled water <br/>
                            &bull; Shaking incubator (INFORS HT) <br/>
-&bull; T4 ligase <br/>
+                   &bull; T4 ligase <br/>
-&bull; T4 buffer<br/><br/>
+                   &bull; T4 buffer<br/><br/>
                         <U>Method:</U><br/>
 . Prepare the following samples in a 1 ml Eppendorf, finishing with the ligase : <br/>
@@ Line 556: / Line 556: @@
                  &bull; Microbiology equipement <br/>
                  &bull; Mastermix 2X <br/>
-                 &bull; Primer S (sense direction)<br/>
+                 &bull; Primer S (sense direction, Forward Primer, see sequence in PCR protocol section)<br/>
-                 &bull; Primers AS (anti-sens<br/>
+                 &bull; Primers AS (anti-sense, Reverse Primer, see sequence in PCR protocol section)<br/>
                  &bull; Phusion DNA polymerase <br/>
                  &bull; DNA template <br/>
@@ Line 563: / Line 563: @@
                  &bull; PCR machine <br/><br/>
              <U>Method:</U><br/>
 . For each insert, make 3 tubes of 0.2 ml with thin walls and prepare the following mastermix : <br/>
                    <table>
 <caption align="bottom" align="center">Table 4</caption>
@@ Line 607: / Line 607: @@
                         </tbody>
                     </table><br/>
-. Distribute the previous mastermix in the tubes and add  template DNA to have : <br/>
+. Distribute the previous mastermix in the tubes and add the template DNA to have : <br/>
 <table>
 <caption align="bottom" align="center">Table 5</caption>
@@ Line 695: / Line 695: @@
 <br/><br/>
 <U>Method:</U><br/>
-. When isopropanol is added, spread the volume in 5x2 2 ml Eppendorf and centrifuge 10 minutes at 13000g <br/>
+. When isopropanol is added, spread the volume in 5 x 2 (2 ml Eppendorfs) and centrifuge 10 minutes at 13000g <br/>
 . Add 1 ml of ethanol in each pellet and centrifuge 10 minutes at 13000g <br/><br/>
 <U>Results:</U><br/>
@@ Line 743: / Line 743: @@
 &bull; PMSF at 100 mM<br/>
 &bull; Cultures of BL21DE3<br/>
-&bull; Buffer B<br/>
+&bull; Buffer A<br/>
 &bull; 50 ml Falcons
 <br/><br/>
@@ Line 749: / Line 749: @@
 .Centrifuge 4x 1l of cultures 20 minutes at 4500g <br/>
 .Throw away the supernatant <br/>
-.Add 40 &#956;l of PMSF in 40 ml of buffer B and add 5 ml of this solution in each pellet <br/>
+.Add 40 &#956;l of PMSF in 40 ml of buffer A and add 5 ml of this solution in each pellet <br/>
 .Scratch the pellet to resuspend it <br/>
-.Add again 5 ml of the solution and mix until it becomes smooth and to recover all the pellet <br/>
+.Add again 5 ml of the buffer A solution and mix the slurry until it becomes smooth, and to recover all the pellet <br/>
 .Put each product in a clean 50 ml Falcon : store at &#8722;80&#176;C. <br/>
 <table>

92. Next step for the growth curve

93. Measurements of insert quantity with Nanodrop

94. Ligation of the new version of the inserts

95. PCR of C1 and C2 (v2)

July 26, 2016:

96. Midiprep of the culture made on the 25^th of July

97. Extraction and resuspension of culture made on the 25^th of July

98. Extraction gel bands C1 and C2

99. Set up the TOPO cloning ratios, ligation of C2 and C1 (v2)

July 27, 2016:

100. STAB culture

101. Electrophoresis of PCR amplifications with the two inserts

102. PCR of the new versions of C1 and C2 at 63 °C

103. Electrophoresis of PCR products from C1 and C2

104. PCR of the new versions of C1 and C2

105. PCR purification

106. TOPO10 transformation with the TOPO cloning ligation product

107. Dosage of C1 v2 and C2 v2 after PCR and purification

July 28, 2016:

108. Send the recombinant vector C2.1 (from the Midiprep of the 26^th of July) for sequencing to Eurofins-MWG

109. Observation of the transformation of C1 v2 and C2 v2 and culture of one colony

110. Preparation of Petri dishes with Xgal

111. Dosage of C1 after PCR product purification

112. Fast Protein Liquid Chromatography (FPLC)

July 29, 2016:

113. SDS PAGE gel of purified protein fractions from FPLC

114. SDS PAGE gel of purified protein fractions from FPLC

115. PCR of C1 v2 and C2 v2

116. Agarose gel Electrophoresis

117. DNA extraction with TOPO cloning colony obtained on the 27^th of July

118. DNA digestion with Xba I and Hind III

July 30, 2016:

119. Protein cryoconservation

Difference between revisions of "Team:Pasteur Paris/Microbiology week8"

Revision as of 16:27, 18 October 2016

click on the weeks

Week 8

July 25, 2016:

Difference between revisions of "Team:Pasteur Paris/Microbiology week8"

Revision as of 16:27, 18 October 2016

click on the weeks

Week 8

July 25, 2016:

92. Next step for the growth curve

93. Measurements of insert quantity with Nanodrop

94. Ligation of the new version of the inserts

95. PCR of C1 and C2 (v2)

July 26, 2016:

96. Midiprep of the culture made on the 25th of July

97. Extraction and resuspension of culture made on the 25th of July

98. Extraction gel bands C1 and C2

99. Set up the TOPO cloning ratios, ligation of C2 and C1 (v2)

July 27, 2016:

100. STAB culture

101. Electrophoresis of PCR amplifications with the two inserts

102. PCR of the new versions of C1 and C2 at 63 °C

103. Electrophoresis of PCR products from C1 and C2

104. PCR of the new versions of C1 and C2

105. PCR purification

106. TOPO10 transformation with the TOPO cloning ligation product

107. Dosage of C1 v2 and C2 v2 after PCR and purification

July 28, 2016:

108. Send the recombinant vector C2.1 (from the Midiprep of the 26th of July) for sequencing to Eurofins-MWG

109. Observation of the transformation of C1 v2 and C2 v2 and culture of one colony

110. Preparation of Petri dishes with Xgal

111. Dosage of C1 after PCR product purification

112. Fast Protein Liquid Chromatography (FPLC)

July 29, 2016:

113. SDS PAGE gel of purified protein fractions from FPLC

114. SDS PAGE gel of purified protein fractions from FPLC

115. PCR of C1 v2 and C2 v2

116. Agarose gel Electrophoresis

117. DNA extraction with TOPO cloning colony obtained on the 27th of July

118. DNA digestion with Xba I and Hind III

July 30, 2016:

119. Protein cryoconservation

96. Midiprep of the culture made on the 25^th of July

97. Extraction and resuspension of culture made on the 25^th of July

108. Send the recombinant vector C2.1 (from the Midiprep of the 26^th of July) for sequencing to Eurofins-MWG

117. DNA extraction with TOPO cloning colony obtained on the 27^th of July