Team:Pasteur Paris/Experiments

Polymerase Chain Reaction

1) PCR Amplification using TaKaRa Ex Taq DNA polymerase

In a 0.2ml tube, set up the following reaction:

	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

Set up the following cycles in a PCR machine
- Initial denaturation: 94°C for 30 sec
- 30 cycles :
- Final extension: 72°C for 5 min.

2) PCR Amplification using Phusion DNA polymerase

In a 0.2ml tube, set up the following reaction:

	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

Set up the following cycles in a PCR machine
- Initial denaturation : 98°C for 30 sec
- 30 cycles :
- Final extension: 72°C for 5 min.

3) PCR Amplification using Q5 High Fidelity Master Mix DNA polymerase

In a 0.2ml tube, set up the following reaction:

	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

Set up the following cycles in a PCR machine
- Initial denaturation : 98°C for 30 sec
- 30 cycles :
- Final extension: 72°C for 2 min.

Enzymatic Digestion

1) Single restriction enzyme digestion

In a MicroCentrifuge tube, set up the following reaction on ice:

	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

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.

2) Double digestion

In a MicroCentrifuge tube, set up the following reaction on ice:

	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

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.

Agarose Gel Electrophoresis

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.

Ligation

Thaw the T4 DNA Ligase Buffer and DNA at room temperature.
Set up the following reaction in a microcentrifuge tube on ice :

	Tube
T4 DNA ligase Buffer (10X)	2µl
Vector DNA (1 fold)
Insert DNA (3 folds)
T4 DNA ligase	1µL
Nuclease free water	to 20 µl
Total	20µl

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.

Transformation in chemically competent E. coli DH5-α

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 1-10ng 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 pre-warmed (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.
Re-suspend 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.

		NB-Esterase	pNP Concentration (mM)
C1	Test	+	50
C1	Test	+	10
C2	Test	+	50
C2	Test	+	10
C3	Test	+	50
C3	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	-

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.

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 competent 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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