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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 -> Sequencing COCHIN
Promoter Characterization Plan
attention le tableau ne correspond pas au titre, j'y travaille... Ana :-)
This table contains the promoters we need to characterize, the transcription factors whose effect on the promoter we want to test, and the plasmid we want to obtain in order to carry out each characterization
| Name
| Ligation
| Biobricks
| Description
|
| MP147.1
| L100.1
| rbs TetR - ECFP
D110 (BV) - D130 (BI)
|    
|
| MP147.2
| L100.2
|
| MP147.3
| L100.3
|
| MP148.1
| L101.1
| rbs TetR - GFP tripart
D110 (BV) - D131 (BI)
|
|
| MP148.2
| L101.2
|
| MP148.3
| L101.3
|
| MP149.1
| L114.1
| AracpBAD - gfp tripart
D126 (BV) - D131 (BI)
| 
|
| MP149.2
| L114.2
|
| MP149.3
| L114.3
|
| MP150.1
| L120.1
| tetR repressible promoter - ECFP
D106 (BV) - D130 (BI)
|
|
| MP150.2
| L120.2
|
| MP150.3
| L120.3
|
| MP151.1
| L122.1
| RBS-lasI - ECFP
D107 (BV) - D130 (BI)
|
|
| MP152.1
| L123.1
| RBS lasI - ECFP
D107 (BV) - D131 (BI)
|
|
| MP151.2
| L123.2
|
| MP152.3
| L123.3
|
| MP153.1
| L126.1
| Strongest RBS (1)- LasR activator (+LVA)
D102 (BV) - D114 (BI)
|
|
| MP153.2
| L126.2
|
| MP153.3
| L126.3
|
| MP154.1
| L132.1
|
|
|
| MP154.2
| L132.2
|
| MP154.3
| L132.3
|
| MP155.1
| L133.1
|
|
|
| MP156.1
| L134.1
|
|
|
| MP156.2
| L134.2
|
| MP156.3
| L134.3
|
| MP157.1
| L138.1
|
|
|
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