Team:Pasteur Paris/Experiments
![](https://static.igem.org/mediawiki/2015/c/c5/Experiments_%26_Protocols_pasteur2015.jpg)
Polymerase Chain Reaction
![](https://static.igem.org/mediawiki/2015/e/ed/IGEM-Pasteur_PCR-PreMix.jpg)
![](https://static.igem.org/mediawiki/2015/4/49/Igem-Pasteur_PCR.jpg)
1) PCR Amplification using TaKaRa Ex Taq DNA polymerase
- In a 0.2ml tube, set up the following reaction:
- Set up the following cycles in a PCR machine
- Initial denaturation: 94°C for 30 sec
- 30 cycles :
- 94°C for 30 sec
- 55°C - 65°C for 1 min depending on your annealing temperature
- 72°C 0.5-1 min per kb
- Final extension: 72°C for 5 min.
|
Tube |
Control |
Control |
Control |
Control |
Ex taq Buffer (10X) |
5 µl |
5 µl |
5 µl |
5 µl |
5 µl |
dNTP mix |
4 µl |
4 µl |
4 µl |
4 µl |
4 µl |
Template DNA |
1-10 ng |
1-10 ng |
1-10 ng |
1-10 ng |
|
Forward Primer |
final concentration: 0.2 µM |
|
final concentration: 0.2 µM |
|
final concentration: 0.2 µM |
Reverse Primer |
final concentration: 0.2 µM |
|
|
final concentration: 0.2 µM |
final concentration: 0.2 µM |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Ex Taq DNA Polymerase |
0.25 µl |
0.25 µl |
0.25 µl |
0.25 µl |
0.25 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
2) PCR Amplification using Phusion DNA polymerase
- In a 0.2ml tube, set up the following reaction:
- Set up the following cycles in a PCR machine
- Initial denaturation : 98°C for 30 sec
- 30 cycles :
- 94°C for 5-10s
- 45°C - 72°C for 10 to 30 sec depending on your annealing temperature
- 72°C for 15-30 sec per kb
- Final extension: 72°C for 5 min.
|
Tube |
Control |
Control |
Control |
Control |
Phusion HF Buffer (5X) |
10 µl |
10 µl |
10 µl |
10 µl |
10 µl |
dNTPs (10mM) |
1 µl |
1 µl |
1 µl |
1µl |
1 µl |
Template DNA |
<250 ng |
<250 ng |
<250 ng |
<250 ng |
|
10µM forward Primer |
2.5 µl |
|
2.5 µl |
|
2.5 µl |
10µM reverse primer |
2.5 µl |
|
|
2.5 µl |
2.5 µl |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Phusion DNA Polymerase |
0.5 µl |
0.5 µl |
0.5 µl |
0.5 µl |
0.5 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
3) PCR Amplification using Q5 High Fidelity Master Mix DNA polymerase
- In a 0.2ml tube, set up the following reaction:
- Set up the following cycles in a PCR machine
- Initial denaturation : 98°C for 30 sec
- 30 cycles :
- 94°C for 5-10s
- 45°C - 72°C for 10 to 30 sec depending on your annealing temperature
- 72°C for 20-30 sec per kb
- Final extension: 72°C for 2 min.
|
Tube |
Control |
Control |
Control |
Control |
Q5 HIgh Fidelity Master Mix (2X) |
25 µl |
25 µl |
25 µl |
25 µl |
25 µl |
Template DNA |
<1 ng |
<1 ng |
<1 ng |
<1 ng |
<1 ng |
10 µM forward Primer |
2.5 µl |
|
2.5 µl |
|
2.5 µl |
10 µM reverse primer |
2.5 µl |
|
|
2.5 µl |
2.5 µl |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
Enzymatic Digestion
1) Single restriction enzyme digestion
- In a MicroCentrifuge tube, set up the following reaction on ice:
- Pipette up and down to homogenize the solution.
- Quick spin in a MicroCentrifuge (5 sec).
- Incubation at 37°C for 1h.
- Heat inactivation for 20 min at 80°C. Optional
- Add the phosphatase: Add 1 unit of Shrimp alkaline phosphatase for each pmol of phosphate end.
- Incubate for 30 min at 37°C.
- Inactivate the phosphatase at 65°C for 15 min.
|
Tube |
Control |
Buffer (10X) |
5 µl |
5 µl |
DNA |
1 µg |
1 µg |
Restriction Enzyme |
1 µl |
|
DNAse, RNAse free water |
to 50 µl |
to 50 µl |
Total |
50 µl |
50 µl |
2) Double digestion
- In a MicroCentrifuge tube, set up the following reaction on ice:
- Pipette up and down to homogenize the solution.
- Quick spin in a Micro-centrifuge (5 sec).
- Incubation at 37°C for 1h.
- Heat inactivation for 20 min at 80°C. Optional
- Add the phosphatase: Add 1 unit of Shrimp alkaline phosphatase for each pmol of phosphate end.
- Incubate for 30 min at 37°C.
- Inactivate the phosphatase at 65°C for 15 min.
|
Tube |
Control |
Control |
Control |
Control |
Buffer (10X) |
5 µl |
5 µl |
5 µl |
5 µl |
5 µl |
DNA |
1 µg |
1 µg |
1 µg |
1 µg |
0 |
Restriction Enzyme n°1 |
1 µl |
|
1 µl |
|
1 µl |
Restriction Enzyme n°2 |
1 µl |
|
|
1 µl |
1 µl |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
Agarose Gel Electrophoresis
![](https://static.igem.org/mediawiki/2015/a/a8/IGEM-Pasteur_illustration-Protocols.jpg)
- Prepare the different dilutions of TAE Buffer (50X, 1X, 0.5X)
- Agarose gel preparation :
- Weigh the agarose (Invitrogen Ref 16500-500) depending on the size of your DNA.
- Dissolve it in the appropriate amount of TAE Buffer (1X).
- Heat the preparation in the microwave until the agarose is dissolved.
- Cool the preparation by letting cool water flow against the Erlenmeyer until there is no evaporation.
- Add 1 drop of EB (Eurobio Ref GEPBET02-AF) under the extraction hood. Mix gently.
- Pour the solution in the casting tray and remove any bubbles.
- Place the combs in the casting tray and let it rest until the gel is solid.
- Carefully remove the combs from the gel
- Place the agarose gel in the electrophoresis apparatus filled with TAE buffer (0.5X).
- Gel loading:
- Load 2 µl of DNA ladder in the first and eventually the last well.
- Load each well with the appropriate amount of DNA and loading buffer.
- Close the electrophoresis unit and run the gel for 1 hour at 130 V and 7 mA.
QIAgen Gel Extraction Kit Protocol
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
- Weigh the gel slice in a colorless tube
- Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes Buffer QG.
- Incubate at 50°C for until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel.
- Check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
- Add 1 gel volume of isopropanol to the sample and mix.
- Place a QIAquick spin column in a provided 2 ml collection tube.
- Apply the sample to the QIAquick column and centrifuge for 1 min.
- Discard flow-through and place the QIAquick column back into the same tube.
- To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min
- Discard flow-through and place the QIAquick column back into the same tube.
- Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g to remove residual wash buffer.
- Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min.
- Let the column stand for 1 min,
- Centrifuge for 1 min.
- If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
![](https://static.igem.org/mediawiki/2015/2/21/IGEM_Pasteur_Gel_extraction_kit.jpg)
Ligation
![](https://static.igem.org/mediawiki/2015/e/e3/Igem-Pasteur_LigationMix.jpg)
- Thaw the T4 DNA Ligase Buffer and DNA at room temperature.
- Set up the following reaction in a microcentrifuge tube on ice :
- Gently mix the reaction by pipetting up and down
- Quick spin in a MicroCentrifuge (5 sec).
- For cohesive ends, incubation at 16°C for 1h.
- Heat inactivation for 10min at 65°C.
|
Tube |
T4 DNA ligase Buffer (10X) |
2µl |
Vector DNA |
|
Insert DNA |
|
T4 DNA ligase |
1µL |
Nuclease free water |
to 20 µl |
Total |
20µl |
Transformation in chemically competent E. coli DH5-α
![](https://static.igem.org/mediawiki/2015/8/84/Igem-Pasteur_TransformationMix.jpg)
- Thaw out on ice one tube of chemically competent E. coli DH5-α.
- Place 50µL of cells in a pre-chilled Micro-centrifuge tube.
- Refreeze any unused cells.
- Add 1-10 ng of DNA to the cells and mix gently by tapping on the tube. Do not mix by pipetting up and down.
- Incubate on ice for 30 min.
- Heat shock the cells for 40 sec in a 42°C water bath.
- Place the tubes on ice for 3 minutes.
- Add 700 µl of prewarmed (37°C) SOC medium (Invitrogen NO 15544-034)
- Incubate at 37°C for 40 min at 200 rpm.
- Spread 200 µl of transformed cells on the appropriate medium.
- Incubate overnight at 37°C
Stab Cultures
- Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar).
- Cool the LB agar to below 50°C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify.
- Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times.
- Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.
- Seal the vial tightly and store in the dark, preferably at 4°C.
MiniPrep
1) Bacterial Culture Growth
- Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic.
- Incubate for 8 h at 37°C with vigorous shaking (~300 rpm).
2) Plasmid purification
- Dilute the starter culture 1/500 to 1/1000 into selective LB medium.
- Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).
- Centrifuge at 6,000 x g for 15 min at 4°C.
- Resuspend the bacterial pellet in 4 ml Buffer P1.
- Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times
- Incubate at room temperature (15–25°C) for 5 min.
- Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times.
- Incubate on ice for 15 min.
- Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
- Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove supernatant containing plasmid DNA promptly.
- Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
- Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
- Wash the QIAGEN-tip with 2 x 10 ml Buffer QC.
- Elute DNA with 5 ml Buffer QF.
- Collect the eluate in a 15 ml or 50 ml tube.
- Add 3.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA.
- Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C.
- Carefully decant the supernatant.
- Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of TE 8.1 Buffer (Tris 10 mM, EDTA 0.1 mM).
MidiPrep
1) Bacterial Culture Growth
- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic.
- Incubate for 12–16 h at 37°C with vigorous shaking.
- Centrifugation at > 8,000 rpm (6,800 x g) in MicroCentrifuge for 3 min at room temperature (15–25°C).
- Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.
2) Plasmid purification
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro-centrifuge tube.
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
- Centrifuge for 10 min at 13,000 rpm in a MicroCentrifuge.
- Apply the supernatants to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30–60 sec. Discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 sec. Discard the flow-through.
- Add 0.75 ml Buffer PE and centrifuge for 30–60 s.
- Discard the flow-through, and centrifuge at full speed for an additional 1 min.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
- Add 50 μl Buffer EB (Qiagen Elution buffer) (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column.
- Let stand for 1 min.
- Centrifuge for 1 min.
Preparation of Electro-competent E. coli BAP 1.
All the following steps take place under sterile conditions and on ice
- Inoculate 500mL of L-broth with 1/100 volume of a fresh overnight E. coli BAP 1 culture.
- Grow the cells at 37 °C at 300 rpm.
- Chill cells on ice for about 20 min.
- Transfer the cells to a cold centrifuge bottle and spin at 4,000 x g for 15 minutes at 4 °C.
- Carefully pour off and discard the supernatant. It is better to sacrifice a few cells than to leave supernatant behind.
- Gently re-suspend the pellet in 500 ml of ice-cold glycerol (10%).
- Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
- Re-suspend the pellet in 250 ml of ice-cold glycerol (10%).
- Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
- Re-suspend the pellet in ~ 20 ml of ice-cold glycerol (10%).
- Transfer to a 30 ml sterile Oakridge tube.
- Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
- Re-suspend the cell pellet in a final volume of 2 ml of ice-cold 10% glycerol. The cell concentration should be about 3 x 1010 cells/ml.
Electroporation of Electrocompetent E. coli BAP 1
- Thaw the cells on ice.
- In a cold, 1.5 ml polypropylene microfuge tube, mix 40 μl of the cell suspension with 5 μl of DNA (DNA should be in a low ionic strength buffer such as TE).
- Mix well and incubate on ice for 1 minute.
- Set the MicroPulser to “Ec1” : for 0.1 cm cuvettes U=1.8 kV and 1 pulse.
- Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom.
- Place the cuvette in the chamber slide.
- Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
- Pulse once.
- Remove the cuvette from the chamber and add 2 ml of SOC medium to the cuvette.
- Quickly but gently re-suspend the cells with a Pasteur pipette.
- Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C for 1 hour, shaking at 225 rpm.
- Plate 200 μL of the cell suspension on a Petri Dish LB+ appropriate antibiotic.
- Incubate overnight at 37 °C.
pNP-Assay
- Preparation of PCS Buffer (1M, pH=7.4)
- 8.0 g of NaCl
- 0.2 g of KCl
- 1.44 g of Na2HPO4
- 0.24 g of KH2PO4
- 1L distilled H2O.
- Preparation of 4-Nitrophenyl butyrate (pNP) in H2O.
- Add 88µL of pNP in 912 µL acetonitrile (ACN) (500 mM)
- Dilute to get 1ml of pNP solution at 1 mM
- Preparation of the Bacterial culture at OD(600 nm)=0.1
- Measure OD(600 nm) of bacterial suspension.
- Dilute the bacterial suspension with PBS buffer.
- In each well of a 96-wells plate (flat-bottom), add 100 µL of bacterial suspension (OD(600 nm)=0.1))
- Incubate at 34°C.
- In each well, add 10 µl of pNP solution.
- For 30 min, OD(405 nm) is recorded every minute for each solution.
|
|
NB-Esterase |
pNP Concentration (mM) |
C1 |
Test |
+ |
50 |
Test |
+ |
10 |
|
C2 |
Test |
+ |
50 |
Test |
+ |
10 |
|
C3 |
Test |
+ |
50 |
Test |
+ |
10 |
|
C1 |
Neg. control 1.a |
+ |
|
C2 |
Neg. control 1.b |
+ |
|
C3 |
Neg. control 1.c |
+ |
|
C- |
Neg. control 2 |
- |
50 |
C- |
Neg. control 3 |
- |
10 |
C- |
Neg. control 4 |
- |
|
Preparation of Electro-competent Saccharomyces cerevisiae cells.
- Inoculate 500 mL of YPD in a 2.8 L Fernbach flask with an aliquot from an overnight culture of Saccharomyces cerevisiae.
- Incubate at 30°C overnight, shaking at 250 rpm.
- Chill the cells on ice water for 15 min to stop growth.
- Decant the cells into to sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C.
- Carefully pour off and discard the supernatant ; place the centrifuge bottles with the cell pellets on ice.
- Add 50 ml of sterile, ice-cold water to each of the bottles and vortex to re-suspend the cell pellets
- Bring the volume in each of the centrifuge bottles to 250 ml.
- Pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C ; pour off and discard the supernatant.
- Wash the cells with a total of 250 ml sterile, ice cold water.
- Re-suspend the cell pellet in 20 ml of sterile, ice cold Sorbitol(1M) and transfer to a chilled 30 ml Oakridge tube.
- Pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C
- Pour off and discard the supernatant.
- Re-suspend the cells pellet in 0.5 mL of sterile, ice cold sorbitol ; the final cell volume should be around 1.3 ml.
Electroporation in Electro-competent Saccharomyces cerevisiae cells.
- Pipette the DNA samples (5-100 ng) to be electroporated into sterile 1.5 mL microfuge tubes. Place tubes on ice.
- Add the competent cells:
- If 0.2 cm cuvettes are used, add 40 µl of the competent cells to each DNA sample
- If 0.4 cm cuvettes are used, add 80 µl of the competent cells to each DNA sample.
- Mix gently and incubate on ice for 5 min.
- Set the MicroPulser to « Sc2 » when using 0.2 cm cuvettes or to « Sc4 » when using 0.4 cm cuvettes. See Section 4 for operating instructions.
- Transfer the DNA-cell samples to the appropriate electroporation cuvettes that have been chilled on ice and tap the suspension to the bottom of the tube.
- Place the cuvette in the chamber slide.
- Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
- Pulse once.
- Remove the cuvette from the chamber and immediately add 1 ml of ice cold sorbitol (1M) to the cuvette. Gently transfer the diluted cells into a sterile tube.
- Check and re-cold the pulse parameters. The time constant should be close to 5 milliseconds.
- Plate aliquots of the electroporated cells on selective agar plates containing sorbitol (1M).
Transformation of SK1 yeast by electroporation
- Inoculate 10 ml of YPD with a yeast strain and grow to saturation at 30°C with shaking.
- Dilute into 40 ml of YPD in a 250 ml or larger sterile flask and grow for 2 hours at 30°C with shaking.
- Collect the cells by centrifugation at 1,000 X g for 5 min.
- Decant the supernatant and re-suspend the pellet in 18 ml of TE Buffer. Then add 2 ml of 1 mM lithium acetate (LiAc).
- Incubate cells at 30°C on a roller drum for 45 min.
- Add 500 μl of DTT (Dithiothreitol)(1M).
- Incubate for 15 min at 30°C on a roller drum.
- Add 80 ml of sterile deionized distilled water (ddH2O) at room temperature.
- Centrifuge cells at 1,000 X g for 5 min.
- Decant the supernatant and re-suspend the pellet in 100 ml of sterile ddH2O.
- Again centrifuge cells at 1,000 X g for 5 min.
- Decant the supernatant and re-suspend the pellet in 5 ml of 1 M Sorbitol.
- Centrifuge cells at 1,000 X g for 5 min.
- Decant supernatant and put cells on ice. Re-suspend the pellet in 120 μl of cold Sorbitol (1M). The volume of resuspended cells should be around 180 μl.
- Keep cells on ice and mix in sterile microfuge tubes
- 40 μl of competent yeast cells
- 1.7 μl of carrier DNA (Salmon sperm DNA)(15 mg/ml)
- DNA to be transformed (up to 5 μl)
making competents cells
- Pre-grow cells in 10 ml YPglu and incubate at 30°C O/N with shaking.
- Dilute to 4x106 cells/ml for S. cerevisiae and to 1.5 .107 cells/ml for C. glabrata in 50 ml YPglu and grow at 30°C with shaking for 3 to 4 h, until 2-3 .107 cells/ml for S. cerevisiae and ~6 .107 cells/ml for C. glabrata (approximately 3 generations). Rq: You will need 1 .108 cells per transformation, so you can adjust the volume of the culture if you planned to do more than 10 transformations for S. cerevisiae or 30 for C. glabrata.
- Harvest enough cells for the number of transformation planned and transfer them in a sterile tube that goes to the centrifuge: 1 .108 cells for one transformation, 2 .108 cells for 2 transformations.
- Centrifuge culture at 4°C, 5 min, 5,000 rpm.
- Wash cells with 20 ml of sterile TE/LiAc.
- Centrifuge at 4°C, 5 min at 5 000 rpm.
- Re-suspend pellet in TE/LiAc in order to have 2 x 109 c/ml : 50 µl per transformation (take into account the volume of the pellet)
- Incubate at 30°C for 15 min without shaking.
Transformation
- Prepare one Eppendorf tube per transformation (include a positive control and a negative control).
- Add 300 µl of TE/LiAc/PEG (made the same day).
- Add 50 µg of carrier DNA (5 µl) that was previously denatured for 3 min at 95°C.
- Add DNA to be transformed (in 1 to 10 µl). Mix with Vortex.
- Add 50 µl of competent cells per tube (108 cells/transformation) and mix carefully with pipetting up and down.
- Incubate at 30°C for 30 min without shaking.
- Heat shock 20 min at 42°C.
- Centrifuge 5 min at 2,000 rpm.
- Re-suspend in 500 µl 5 mM CaCl2 and incubate at RT for 5-10 min.
- Centrifuge 5 min at 2,000 rpm and re-suspend in H2O.
- Spread cells with glass beads on selective media, 1/100, 1/10 or more of the transformation.
^
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