Team:Paris/Notebook/Protocols

Extraction

• For each new extraction it's important to have a new bath of ETB
• Use a new blade for each extraction
• The band weight must be less than 200 mg

Amplification of promoters

=>To amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

• Preparation of the templates : Resuspend of 1 colony in 100µl of water.
• Preparation of PCR mix :

For each samples, 1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)

• make a mix with buffer, oligos and water for n+1 samples
• negative control : without template
• positive control : known template

• Program PCR : PROMOTEU

LID : 105°C
1. 95°C 5 min
2. 95°C 1 min denaturation
3. 60°C (depending of the size of oligos) 30 sec annealing
4. 72°C (1 min for 1 kb)
5. go to : 2 rep : 24-29
6. 72°C 5 min
7. sound : 1
8. hold : 10°C

Purification (Kit Promega)

Gel Slice and PCR Product Preparation

Dissolving the Gel Slice

• Following electrophoresis, excise DNA band from gel slice in a pre-weighed 1.5 mL microcentrifuge tube.
• Add 10µL membrane Binding Solution per 10 mg of gel slice. We prefer not to vortex and we incubate at 50-65°C until gel slice is completely dissolved (∼10 min). Quick centrifuge.

Processing PCR reactions

For products above 40 pb

• Add an equal volume of Membrane Binding Solution to the PCR reaction.

Binding of DNA

• Insert the SV Minicolumn into Collection Tube.
• Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
• Centrifuge at 16,000 x g for 1 minute. Discard the flowthrough and reinsert Minicolumn into Collection Tube.

Washing

• Add 700 µL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert the Minicolumn into Collection Tube.
• Repeat Step 4 with 500 µL Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes.
• Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Elution

• Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube.
• Add 30 µL Buffer EB (Qiagen). Incubate at room temparature for 1 minute. Centifuge at 16,000 x g for 1 minute.
• Discard Minicolumn and store at 4 or -20°C.

Quantification by electrophoresis

• Gel : 1.5-2% agarose with BET added (5 µL BET for 100 mL TBE)
• 10 µL Quick-Load 1 kb DNA Ladder
• 2 µL LB + 3 µL DNA

Ligation

• 2 µL Ligase Buffer 10X
• X µg/µL vector
• 3 or 4 x X µg/µL insert
• Pure water qsp 20 µL
• 1 µL T4 ligase
• O/N at 16°C

Transformation

Use of TOP10 chemically competent cells

• Defroze competent cells on ice during 5'
• Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
• Incubate 30' on ice
• Heat-shock the cells during 30" at 42°C without shaking
• Put 2' on ice
• Add 250µL of pre-warmed SOC medium (42°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Spin at 5.000rpm during 30"
• Remove 150µL of supernatant
• Resuspend the pellet in the 150µL left
• Spread on adequated plates
• Incubate O/N at 37°C

PCR Screening

Use of 8 clones of Ligation transformants for screening PCR

• One toothpick of each clone's colony per PCR tube
• Use toothpick to start 7.5mL O/N culture

After, add

• 25µL Mix
• 1µL Oligo F (10µM)
• 1µL Oligo R (10µM)
• 23µL pure water
• negative control : without clone's colony
• positive control

• Program : SCREENIN

LID 105°C
1. 95°C 5min
2. 95°C 30 sec
3. 55°C 30 sec
4. 72°C (1 min for 1kb)
5. go to : 2 rep : 29
6. sound : 1
7. hold : 4°C

Electrophoresis Purification of PCR

• 10µl of ladder 100 pb or 1 kb
• 4 µl of screening PCR
• Migration at 100V on 1,5% gel until 3/4 way

Sequencing