Team:Paris/Notebook/Protocols

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Revision as of 15:53, 11 August 2008

Culture of Stable strain with biobricks 2008

Streaks on plates with LB and the adadpted antibiotics to isolate colonies
Incubate O/N at 37°C

Take clone with a toothpick and put in 7.5ml LB with adaptated antibiotics
Will be use for Miniprep and Stock in glycerol
2-3 clones isolated by Biobricks
Incubate O/N at 37°C

Glycerol Stocks

Remove 2.5mL of each culture and centrifuge.
Discard the supernatant and resuspend pelleted bacterial in 1mL of LB.
Add 500µL of 60% glycerol.
Stored at -20°C.

Minipreps (Kit Qiagen)

centrifuge 5mL of culture 8 min at 3,500 to 4,000 g.
Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue.
Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30–60 s. Discard the flow-through.
Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details)
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 30 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Electrophoresis

An electrophoresis can be done to check if there is Product of Miniprep

Gel : 1% 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

Concentration of the Miniprep

By biophotometry

Blank : 60 µL of pure water
Sample : 50 µL of pure water + 5 µLof DNA

Check if the ratio 260/280 is over 1,6
!!!Think about the dilution!!!

Digestion

1 µg of plasmid
Buffer (n°2) 10X
BSA 100X
Pure water qsp 30 µL
1 µL of each enzyme

Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).

Migration after digestion for vectors

Run the whole samples (30 µL) in a 0.8-1% agarose gel (a new one)
Run at 50 W until halfway
10 µL of ladder 1kb and 100 pb on every side
30 µL of DNA + 6 µL of LB

!!!Separate each band by an empty one!!!

Extraction

For each new extraction it's important to have a new bath of BET
Use a new blade for each extraction
The band weight must be under 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 competentcells

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
Resuspent the pellet in the 150µL left
Spread on adequated plates
Incubate O/N at 37°C

Screening PCR

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 100W on 1,5% gel until 3/4 way