Team:GO Paris-Saclay/labnotebook-protocols

Component	Volume (uL) for 25 uL reaction	Volume (uL) for 50 uL reaction
Template DNA (up to x ng)	Variable	Variable
10 X Green Taq buffer	2, 5	5
2, 5 mM dNTPs	2	4
FW Primer	1, 25	2, 5
BW Primer	1, 25	2, 5
Taq polymerase	0, 1	0, 5
Nuclease-free H2O	18, 1	Up to 50 uL

Add the total volume per sample (without DNA) to each PCR tube.
Add the template DNA to each tube.
Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):

Step	Temperature (°C)	Time	Cycle
Initial denaturation	95	3 min	1
Denaturation	95	30 s	30
Annealing	Variable	30s
Extension	72	1 min/kb
Final extension	72	5 min	1
Hold	4	∞

first 5 cycles with annealing temperature 45 °C, extension 40s, then 30 cycles with annealing temperature 55°C, extension 40s.

Run a DNA electrophoresis following the DNA electrophoresis protocol.

DNA electrophoresis

Pour the solution Agarose (0, 8%)-EtBr into the mould. Add a comb to create wells for the samples. Let it solidify (approx. 20 minutes).
Transfer the gel to the electrophoresis Remove the combs and cover it with TAE (0, 5 X). Transfer the gel to the electrophoresis
Load the molecular weight marker (ladder) in the first well (check the appropriate volume for each marker, generally 5 µL works fine) and load 5-10 µL of the samples in the other wells.
Run at 100 V for 25 min

PCR product purification (Nucleospin Gel and PCR Clean-up)

NB: Before starting check if the buffers are prepared like indicated in the kit protocol

Mix 1 volume of sample with 2 volumes of Buffer NTI
To bind the DNA place a NucleoSpin® PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample.
Centrifuge for 30 s at 11,000 xg
Discard flow-through and place the column back into the collection tube. Load remaining sample if necessary and repeat the centrifugation step.
To wash silica membrane, add 700 μL Buffer NT3to the column.
Centrifuge for 30 s at 11,000 x g.
Discard flow-through and place the column back into the collection tube.
To dry the silica membrane centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
To elute DNA place the column into a new 1.5 mL microcentrifuge tube.
Add 15–30 μL Buffer NE and incubate at room temperature for 1 min
. Centrifuge for 1 min at 11,000 x g

DNA concentration measurement (NanoDrop)

Turn on the NanoDrop
Press the button for dsDNA to measure the concentration of double-stranded DNA in your samples.
Clean the measurement surface with a piece of tissue and ethanol.
Use the elution buffer or the media where your DNA is contained as a blank.
Clean the measurement surface with a piece of tissue and water.
Use 2 µL of sample to measure its concentration.
If you have multiple samples, clean the measurement surface in between measurements.

Heat-shock Transformation

Start the water bath at 42°C
Let the competent cell thaw on ice
Add 1 µL of plasmid (or 5 µL of ligation mix) to the tube and flick gently
Let the tube on ice for 30 min
Put the tube in water (42°C) 1 min
Put the tube on ice for 5 min
Add 500 µL of LB to the tube
Leave the tube to shake at 37°C for 1 h 30 min.
Plate 50 µL on a LB plate with appropriate antibiotics.

Plasmid extraction (Mini and Midi Prep)

Mini Prep

Liquid culture (5 mL)

Label as many 15 mL hemolysis tubes as the number of colonies you want to grow.
Prepare 5 mL of selective medium (LB) per hemolysis tube.
Under sterile conditions, pick a colony with the sterile toothpick.
Swirl the toothpick in the hemolysis tube containing the medium. Often you can see the colony falling from the loop.
Grow the liquid cultures to shake overnight, at 37 °C.
To conserve this liquid culture, glycerol 60% can be added to the bacteria culture and the cryotubes are put at -20°C.

Digestion assay

Decide on which enzyme(s) to cut with. Check online what buffer the enzyme(s) work(s) in. For Bio labs enzyme use the Double Digest Finder.
Prepare a sample as follows:

Compound	Volume (µL)
DNA (plasmid or insert	Variable according to the number of base and the concentration
Buffer (NEB, FD, SmartCut)	2
Restriction enzyme 1	1
Restriction enzyme 2	1
Water	Up to 20 µL

Incubate for one hour at 37 °C.

Gel product purification (Nucleospin Gel and PCR Clean-up)

Excise DNA fragment from the gel
Add 200 uL buffer NT1 foreach 100mg of agarose <2%. For gels containing > 2% agarose, double the volume of NT1
Incubate sample for 5–10 min at 50 °C
Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved.
To bind the DNA place a NucleoSpin® Gel Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample.
Centrifuge for 30 s at 11,000 xg
Discard flow-through and place the column back into the collection tube. Load remaining sample if necessary and repeat the centrifugation step.
To wash silica membrane, add 700 μL Buffer NT3 to the column.
Centrifuge for 30 s at 11,000 x g.
Discard flow-through and place the column back into the collection tube.
To dry the silica membrane centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
To elute DNA place the column into a new 1.5 mL microcentrifuge tube.
Add 15–30 μL Buffer NE and incubate at room temperature for 1 min
. Centrifuge for 1 min at 11,000 x g

DpnI digestion

Ligation assay

Prepare the mix for all the samples in a single 1.5 mL tube. For one sample:

Compound	Volume (uL)
Insert	Variable according to the number of base and the concentration
Plasmid	Variable according to the number of base and the concentration
T4 buffer 10 X	1
T4 ligase	1

Wait one hour until ligation

Plasmid DNA extraction (Mini, midi and maxi Prep)

MiniPrep

Use 1–5 mL of a saturated E. coli LB culture, pellet cells in a s microcentrifuge for 30 s at 11,000 x g
Discard the supernatant and remove as much of the liquid as possible.
Add 250 μL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down.
Add 250 μL Buffer A2. Mix gently by inverting the tube 6–8 times. Do not vortex to avoid shearing of genomic DNA
Incubate at room temperature for up to 5 min or until lysate appears clear.
Add 300 μL Buffer A3. Mix thoroughly by inverting the tube 6–8 times until blue samples turn colorless completely.
Centrifuge for 5 min at 11,000 x g at room temperature. Repeat this step, in case the supernatant is not clear.
Place a NucleoSpin® Plasmid / Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 750 μL of the supernatant onto the column.
Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the column into the collection tube.
Repeat this step to load the remaining lysate.
Add 500 μL Buffer AW, preheated to 50 °C
Centrifuge for 1 min at 11,000 x g
Add 600 μL Buffer A4
Centrifuge for 1 min at 11,000 x g.
Discard flowthrough and place the column back into the emptycollection tube.
Centrifuge for 2 min at 11,000 x g and discard the collection tube.
Place the column in a 1.5 mL microcentrifuge tube and add 50 μL Buffer AE. Incubate for 1 min at room temperature.
Centrifuge for 1 min at 11,000 x g.

Midi and MaxiPrep (Qiagen)

Pellet 25 mL or 100 mL (high copy) or 100 mL, or 500 mL (low copy) overnight LB culture at

4 700 rpm for 30 min at 4°C.

Resuspend the bacterial pellet in 4 mL or 10 mL buffer P1.
Add 4 mL or 10 mL buffer P2, mix thoroughly by inverting 4-6 times, and incubate at room temperature for 5 min.
Add 4 mL or 10 mL buffer P3, mix thoroughly by inverting 4-6 times, and incubate at room temperature for 15 or 20 min.
Centrifuge at 4 700 rpm for 50 min at 4°C. Re-centrifuge the supernatentat 4 700 rpm for 25 min at 4°C.
Equilibrate a Qiagen -tip 100 or 500 by applying 4 mL or 10 mL buffer QBT and allow column to empty by gravity flow.
Apply the supernatant (step 5) to the tip and allow it to enter the resin by gravity flow.
Wash the tip with 2 X 10 mL or 2 x 30 mL buffer QC. Allow the buffer QC to move through the tip by gravity flow.
Elute DNA with 5 mL or 15 mL buffer QF into clean 15 mL or 50 mL vessel.

PCR with Q5

Prepare the mix for all the samples in a single 1.5 mL tube. For one sample:

Component	Volume (uL) for 25 uL reaction
Template DNA (up to x ng)	0,5
5X Q5 buffer	5
2, 5 mM dNTPs	2
FW Primer	1, 25
BW Primer	1, 25
Q5 polymerase	0, 25
Nuclease-free H2O	15, 25

Add the total volume per sample (without DNA) to each PCR tube.
Add the template DNA to each tube.
Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):

Step	Temperature (°C)	Time	Cycle
Initial denaturation	98	30 s	1
Denaturation	98	10 s
Annealing	Variable	30s	30
Extension	72	30s/kb
Final extension	72	2 min	1
Hold	4	∞

xcPCR phusion

Prepare the mix for all the samples in a single 1.5 mL tube. For one sample:

Component	Volume (uL) for 50 µL reaction
Template DNA (up to x ng)	1
5X buffer HF	10
2, 5 mM dNTPs	4
FW Primer 10 uM	2, 5
BW Primer 10 uM	2, 5
Phusion polymerase	0, 5
Nuclease-free H2O	29, 5

Add the total volume per sample (without DNA) to each PCR tube.
Add the template DNA to each tube.
Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):

Step	Temperature (°C)	Time	Cycle
Initial denaturation	98	30 s	1
Denaturation	98	10 s
Annealing	Variable	30s	30
Extension	72	30s/kb
Final extension	72	5 min	1

Phage transduction

Phage lysat

Add CaCl2, final concentration 5mM to overnight bacterial culture
Prepare 3 tubes (1, 5 mL) per lysate:

0,1 ml of bacteria + 30 µl of the P1vir phage (~ 106 Φ/µl)
0,1 ml of bacteria + 5 µl of the P1vir phage (~ 106 Φ/µl)
0,1 ml of bacteria (negative control tube)

Incubate 10- 20 min at room temperature (without agitation).
Add 1 ml of LB per tube.
Add 3 ml of melted Top Agar in a hemolysis tube to the bacteria/phage suspension. Be careful not to kill the bacteria with the heat of the melted Top Agar
Spread the bacteria/phage suspension on LCA + CaCl2 (5.10-3 M) petri dishes –
Incubate 3 to 6 hours at 37°C, until the culture turns colorless.
Reap it off and place it in a 15 mL falcon tube. If needed, you may leave the dishes overnight at 30°C before collecting the mat.
Add 1 mL of chloroform and vortex the tube.
Centrifuge 10 min at 4000 x g.
Recover the supernatant.