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Team:Bielefeld-CeBiTec/Notebook/Protocols
From 2014.igem.org
Protocols
- Pick one colony with a sterile tip and elute it in 100 µL ddH20 or medium
- Store the colony in 4 °C while colony PCR is running
- One reaction mix contains:
- 5 µL 5x buffer
- 1 µL MgCl2 (25 mM stock)
- 0.5 µL dNTPs
- 0.25 µL primer mix (prefix/suffix primers or sequencing primers)
- 17.625 µL ddH2O
- 0.125 µL GoTaq polymerase (Promega)
- 0.5 µL template
- PCR program:
- Cell lysis and initial denaturation: 5 min, 95 °C
- 30 cycles of:
- 10 s, 95 °C
- 30 s, annealing temperature
- 1 min / 1 kb of expected product, 72 °C
- Final elongation: 5 min, 72 °C
- Gel electrophoresis: check the fragment size
- Plate the correct colony
- Pick one colony with a sterile tip and streak cells at a marked position on a new plate
- Put tip in PCR tube already containing the reaction mixture
- One reaction mix contains:
- 3 µL 5x buffer
- 0.5 µL MgCl2 (25 mM stock)
- 0.3 µL dNTPs
- 1 µL of each primer (for example prefix/suffix primers or sequencing primers)
- 9 µL ddH2O
- 0.08 µL GoTaq2 polymerase (Promega)
- PCR program:
- Cell lysis and initial denaturation: 5 min, 95 °C
- 40 cycles of:
- 10 s, 95 °C
- 30 s, annealing temperature
- 1 min / 1 kb of expected product, 72 °C
- Final elongation: 5 min, 72 °C
- Gel electrophoresis: check the fragment size
- Use cells from the right positions to start liquid cultures or streak them on a new plate
- Material:
- 550 mL LB-Medium
- 1 L cooled bidest. H2O
- 150 mL cooled 10 % glycerine
- 10 pre-cooled 50 mL Falcons
- Protocol:
- Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
- Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
- Incubate until OD600 0,4-0,6
- Cool the culture 15-30 minutes on ice
- Onwards all steps at 4°C
- Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
- Discard supernatant
- Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
- Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
- Discard supernatant
- Resuspend pellet in 5 mL cooled bidest H2O
- Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
- Discard supernatant
- Resuspend pellet in 5 mL cooled 10 % glycerine
- Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
- Discard supernatant
- Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
- Divide cells in 50 µL aliquots and freeze in liquid N2 immediately
- Store at -80 °C
- Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
- Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
- Store cells on ice for 1 minute
- Electroporate at U = 2.5 kV, C = 25 µF, R = 400 ?
- Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
- Centrifuge 1 second at maximal rpm and plate on selective LB-Medium
- Incubate over night at 37 °C
- Dissolving the Gel Slice
- Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube.
- Add 10 µL Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65°C until gel slice is completely dissolved.
- Processing PCR Amplifications
- Add an equal volume of Membrane Binding Solution to the PCR amplification.
- Binding of DNA
- Insert 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 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 Minicolumn into Collection Tube.
- Repeat Step before 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 minute 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 15 µL of Nuclease-Free Water to the Minicolumn. Incubate at 60°C for 5 minutes. Centrifuge at 16,000 x g for 1 minute. Repeat this step.
- Discard Minicolumn and store DNA at 4°C or -20°C.
- Note:
- All purification steps should be carried out at room temperature.
- All centrifugation should be carried out in a table-top microcentrifuge at >12000 x g
(10 000-14 000 rpm, depending on the rotor type). - Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
- Note Ensure RNase A has been added to the Resuspension Solution.
- Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
- Note Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 minutes to avoid denaturation of supercoiled plasmid DNA.
- Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
- Note It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudly.
- Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.
- Transfer the supernatant to the supplied GeneJET spin coloumn by decanting or pipetting. Avoid disburbing or transferring the white precipitate.
- Centrifuge for 1 minute. Discard the flow-through and place the coloumn back into the same collection tube.
- Note Do not add bleach to the flow-through.
- Add 500 µL of the Wash Solution (diluted with ethanol prior to first use) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
- Repeat the wash procedure (step before) using 500 µL of the Wash Solution.
- Discard the flow-through and centrifuge for an additional 1 minute to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
- Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmide DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 minutes at room temperature and centrifuge for 2 minutes.
- Note An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
- Discard the column and store the purified plasmid DNA at -20°C.
