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Culture of Stable strain with biobricks 2008
- Streaks on plates with LB and the adapted 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 bacteria in 1mL of LB.
- Add 500µL of 60% glycerol.
- Store 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 / 250 ng of gene
- Buffer (n°2) 10X : 3µL
- BSA 100X : 0.3µL
- 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 V 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 | !
- 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
LID : 105°C
1. 98°C 30 sec initial denaturation
2. 98°C 10 sec 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
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
voir ici -> [http://institut.cochin.inserm.fr/rubric_recherche/Plates-Formes/sequencage_genomique/I18NFolder.2005-02-10.4781618697/page2/fr Sequencing COCHIN]
Promoter characterization plan
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