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)
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)
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. TOP 10 cells transformation with the TOPO cloning ligation product
107. Dosage of C1 v2 and C2 v2 after PCR and purification
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:
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:
| Times | OD600nm |
|---|---|
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 |
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
| 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 |
A260 ⁄ 280 |
1.11 | 1.20 | 2.35 | 2.97 | 2.76 | 3.16 | 2.35 | 3.04 | 2.79 | Ø |
A260⁄230 |
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 :
| 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 |
Vfinal (μl) |
15 | 15 | 15 | 15 |
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 :
| 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 |
| 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 |
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)
| 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 1 l of cultures 20 minutes at 4500g, in a Beckman JLA 8.1000 rotor in a Avanti x26-J centrifuge
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.
| 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 :
| 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 |
| C1 | C2 | |
|---|---|---|
OD260 nm |
0.003 | 0.003 |
OD280 nm |
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:
| C1 v2 | C2 v2 | |
|---|---|---|
DNA from colonies (inserts) (μl) |
4 | 4 |
Salt solution (μl) |
1 | 1 |
Distilled water (μl) |
5 | 5 |
TOPO vector (μl) |
1 | 1 |
Vfinal (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 mg⁄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, OD600 nm of approx. 0.7) and check the concentration at several times during it :
| Time | OD600 nm |
|---|---|
0 |
0.007 |
1h19 |
0.059 |
2h39 |
0.458 |
3h54 |
0.827 |
Aim: Separate the amplification bands of specific DNA (900 bp) from non-specific bands (700 bp).
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 :
| 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 |
| 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 |
Aim: Check the results of the previous PCR of the new versions of C1 and C2
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:
| 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 |
Figure 8 : PCR gel of C1/C2
Aim: PCR of the new versions of C1 and C2 at lower annealing temperature 55 ºC
Re-amplify some of the previous PCR reaction solution with fresh dNTPs, buffer and polymerase..
Protocol: follow this link
What we did in the lab:
Materials:
• Samples from the previous PCR of the 26⁄07
• Tubes for PCR
• PCR machine
• dNTPs
• 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: dNTPs, MgCl2 and Phusion enzyme :
| Volume (μl) | Insert | Primer S | Primer AS | Without DNA |
|---|---|---|---|---|
Samples |
45 | 45 | 45 | 45 |
dNTPs |
6 | 6 | 6 | 6 |
MgCl2 |
8.4 | 8.4 | 8.4 | 8.4 |
Phusion |
0.5 | 0.5 | 0.5 | 0.5 |
Vfinal |
60 | 60 | 60 | 60 |
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) = 165 mg which correspond to 496.5 µl of buffer
4. Follow the QIAGEN gel extraction Kit protocol
Results :
Figure 9 : PCR gel of C1/C2
Aim: Transform our new plasmid (vector TOPO + C1 v2 and C2 v2).
Protocol: follow in this link
What we did in the lab:
Materials:
• Microbiological equipment
• Carbenicillin at 50 mg⁄ml
• LB medium
• Petri dish
• TOP 10 competent cells
• Dimethylformamide (DMF)
• Xgal 20 mg/ml in DMF
Method:
1. Prepare the solution of Xgal for the Petri dish with :
1.a 10 ml of DMF
1.b 200 mg of Xgal
2. Put 100 µl of this solution on 6 Petri dishes which already contain LB and carbenicillin. Then spread the solution in an additional volume of LB (100 &181#l). Let the Petri dishes dry at 37°C for approx. 1 hr.
3. Recovery C2 v2 and C1 v2 prepared on the 26⁄07 (to perform the TOP 10 cloning reaction)
4. Put 50 µl of bacteria TOP 10 in ice and add 6 µl of C1 v2 or C2 v1
5. Follow the protocol to transform thermally competent bacteria
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 TOP 10 cells.
What we did in the lab :
Materials :
• Solution from TOP 10 transformation with the ligation TOPO10 cloning product (experiment from the 27⁄07)
•DMF
• 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 DMF and 10 mg of Xgal
3. Spread 200 µl of Xgal on the Petri dishes, and allow drying at 37°C for approximately 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 :
• Ultrospec 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
| OD260 | OD280 | [DNAC1] (µg⁄ml) |
|---|---|---|
| 0.002 | 0.001 | 50 |
Aim: Protein purification.
Protocol: follow in this link
What we did in the lab :
Materials :
• FPLC machine (Fast Protein Liquid Chromatography)(Akta Prime Plus, GE Lifesciences)
• 5 ml Ni-NTA His-trap column (GE lifesciences)
• Buffer A (Tris 50 mM pH 7.4 ; NaCl 150 mM), Buffer B=Buffer A + Imidazole 250 mM
• PMSF at 100 mM in ethanol
• 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 the FPLC machine with buffer A
3. Once the bacteria are 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 30 seconds of sonication followed-up with 1 minute 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 X26-J at 4°C during 30 minutes
9. Transfer the supernatant to 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 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% to 100% buffer B over 12 column volumes and collect fractions of 1 ml
15. Wash the column with buffer A
Aim: Detection of the proteins present 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 SDS)
• Electrophoresis chamber , Power supply and electrodes (Biorad)
• 2X SDS (Laemli buffer with β-mercapto ethanol)
• 95°C Heater block
• Gel Code blue stain (Pierce) with G250 Coomassie blue
Method
1. At the beginning, prepare 1X from 10X TGS buffer with 25 ml of 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 : Well 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 10 : SDS PAGE gel of our protein
| Fractions from FPLC | Dilution | Contain |
|---|---|---|
| 19, 21, 25 | Ø | Ø |
| 30, 34, 36 | (1/100) in buffer A | 1 µl of protein with 99 µl of buffer A |
| 37, 38, 39 | (1/10) in buffer A | 1 µl of protein with 9 µl of buffer A |
Figure 4 : SDS PAGE gel of diluted proteins
| Volumes (µl) | |
|---|---|
| dNTPs |
22.5 |
| MgCl2 |
22.5 |
| RNA free |
327.6 |
| Buffer 10X pfu |
22.5 |
| Total |
417.6 |
| Tubes | Name | V(premix) (µl) | V(C1 v2) (µl) | V(C2 v2) (µl) | V(primer For) (µl) | V(primer Rev) (µl) | V(H2O) (µl) |
|---|---|---|---|---|---|---|---|
| 1 |
C1 v2 insert For and Rev | 46.4 | 1 | 0 | 1 | 1 | 0 |
| 2 |
C1 v2 insert For | 46.4 | 1 | 0 | 1 | 0 | 1 |
3 |
C1 v2 insert Rev | 46.4 | 1 | 0 | 0 | 1 | 1 |
| 4 |
C2 v2 insert For and Rev | 46.4 | 0 | 1 | 1 | 1 | 0 |
1 |
C2 v2 insert For | 46.4 | 0 | 1 | 1 | 0 | 1 |
1 |
C1 v2 insert Rev | 46.4 | 0 | 1 | 0 | 1 | 1 |
| 1 |
Without DNA | 0 | 0 | 0 | 0 | 0 | 0 |
| 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 |
Figure 5 : PCR gel of C1 v2 / C2 v2
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 :
| Samples | A260 nm | A280 nm | [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
| Volumes (μl) | |
|---|---|
DNA (10 μg⁄ml) |
5 |
Xba I |
1 |
Hind III |
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 = 32 ml
