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Team:Paris/Notebook/Protocols
From 2008.igem.org
Culture of Stable strain with biobricks 2008
- In 6ml LB with adaptated antibiotics
- Will be use for Miniprep and Stock in glycerol
- 2 clones isolated by Biobricks
- O/N at 37°C
Glycerol Stocks
- 1mL of each culture (with 2 clones) has been added to 1mL of 40% glycerol.
- For each clone, two glycerol stocks have been done.
- Stored at -20°C.
Minipreps (Kit Qiagen)
- 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 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
Qualitative and quantitative analysis by electrophoresis
- Gel : 1% agarose
- 10 µL Quick-Load 1 kb DNA Ladder
- 2 µL LB + 3 µL DNA
- Bath of BET 20000X (5 µL BET for 100 mL TBE) during about 5 min.
=> Comparing the concentration of the miniprep thanks to the ladder
Check if the mesured size corresponds with the expecting size.
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!!!
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)
- Program PCR : PROMOTEU
LID : 105°C
1. 95°C 5 min
2. 95°C 1 min
3. 60°C 30 sec
4. 72°C 1 min 30 sec
(1 min for 1 kb)
5. go to : 2 rep : 29
6. sound : 1
7. hold : 10°C
Digestion
- 1 µg of plasmid (Miniprep)
- Buffer (n°2) 10X
- BSA 100X
- Pure water qsp 30 µL
- 1 µL of each enzyme
- Incubate during about 3h at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).
Migration after digestion
Separation of insert and vector
- Run the whole samples in a 1,5% agarose gel
- About 30 minutes at 100 W
- 10 µL of ladder 1kb and 100 pb on every side
- 3 µL of DNA + 2 µL of LB
!!!Separate each band by an empty one!!!
For promoters
- 1,5% agarose gel
- 10 µL of ladder 100 pb
- 3 µL of digestion product
- 2 µL of LB
- Run at 100 W about 30 min
For vectors
- 1% agarose gel (a new one)
- 10 µL of ladder 1 kb
- whole of digestion product
- 2 µL of LB
- Run at 50 W about 30 min
Extraction
- For each new extraction it's important to have a new bath of BET
- Use a new blade for each extraction
- The extraction must be under 400 mg
Purification (Kit Promega)
Gel Slice and PCR Product Preparation
Dissolving the Gel Slice
- Following electrophoresis, excise DNA band from gel slice in a 1.5 mL microcentrifuge tube.
- Add 10µL membrane Binding Solution per 10 mg of gel slice. We prefer do not vortex and we incubate at 50-65°C until gel slice is completely dissolved (∼10 min). Centrifuge.
Processing PCR reactions
For products under 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.
Ligation
- 2 µL Ligase Buffer 10X
- X µL insert
- X/3 µL vector
- 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 (4°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 by tube
After, add
- 25µL Mix
- 1µL Oligo F (10µM)
- 1µL Oligo R (10µM)
- 23µL pure water
- Program : SCREENIN
LID 105°C
1. 95°C 5min
2. 95°C 30 sec
3. 55°C 30 sec
4. 72°C 1 min 30 sec
5. go to : 2 rep : 29
6. sound : 1
7. hold : 4°C
