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Team:TU Darmstadt/Protocols/Metabolism
From 2012.igem.org
Protocols Metabolism
The following page give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012.
Contents |
In vivo
Production of chemically competent cells
Chemically competent cells are needed for transformation with the heat shock method. We used CaCl2 to produce them.
Production of chemically competent cells
- Inoculate 10 ml of LB-Media with an E. coli colony and incubate at 37 °C overnight
- Inoculate 200 ml of LB-Media with 2.5 ml of the overnight culture
- Incubate the culture at 37 °C until an OD600 of 0.5
- Cool the cells on ice for 5 min
- Centrifuge the cells for 10 min at 5000 rpm and 4 °C
- Resuspend the cell-pellet in 32 ml pre-cooled CaCl2 solution (Do not vortex!)
- Add pre-cooled CaCl2 solution to 200 ml
- Let the cells repose on ice for 1 hour
- Centrifuge the cells for 10 min at 5000 rpm and 4 °C
- Resuspend the pellet in 10 ml cryo-solution
- Decant 200 µl of competent cells in a 1.5 ml tube
- Store the tube in an -80 °C freezer
Solutions
- CaCl2
- 5.55 g CaCl2
- Add di H2O to 1 L
- Sterilize by autoclaving
- Cryo solution
- 0.278 g CaCl2
- 10 ml glycerin
- Add di H2O to 50 ml
- Sterilize by autoclave
Heat shock transformation with E. coli
- Thaw the chemically competent cell on ice
- Add 50 – 300 ng of the purified plasmid or 1-5 µL of the heat inactivated ligation mix to the cells (10 – 500 ng DNA)
- Invert the tube up to 6 times
- Incubate the cells on ice for 30 min
- Incubate the cells for 1 min at 42 °C
- Let the cells cool down on ice for 5 min
- Add 800 µl of SOC medium
- Incubate the cells for 45 min at 37 °C
- Centrifuge for 5 min at 0.4 rcf
- Resuspend the pellet in 100 µl LB-Media and plate it on LB Agar with antibioticum
Glycerine stock
- Add 200 µl of sterilized glycerine to 800 µl cell culture and mix well
- Freeze the stock at -20 °C
In vitro
PCR
The Polymerase Chain Reaction (PCR) is used to amplify DNA from bacterial colonies or DNA templates.
Colony PCR (isolation of genomic DNA)
- Pick one colony with a sterile tip
- One reaction mix contains:
- 10 µL of 5x Phusion HF Buffer
- 1 µL of dNTPs (10 mM each)
- 0,5 µL of Phusion High-Fidelity Polymerase
- Forward primer (10 pmol)
- Reverse primer (10 pmol)
- 1,5 µL of DMSO
- DI water to 50 µL
- Colony template
- PCR program
# Temperature Time 1 98 °C 00:02:00 2 Ta 00:01:00 3 72 °C 00:01:00 4 98 °C 00:01:00 5 Ta 00:01:00 6 72 °C 00:01:00 7 GO TO 4 REPEAT 31x 8 98 °C 00:01:00 9 Ta 00:01:00 10 72 °C 00:06:00 11 4 °C HOLD
Colony PCR (verification of transformation)
- Pick one colony with a sterile tip and suspend in 10 µL of DI H2O
- One reaction mix contains:
- 2 µL of 10x Thermopol Reaction Buffer
- 0,4 µL of dNTPs (10 mM each)
- 0,3 µL of Taq DNA Polymerase
- VF2 (10 pmol)
- VR (10 pmol)
- 0,6 µL of DMSO
- 1 µL of colony suspension
- DI water to 20 µL
- PCR program
# Temperature Time 1 95 °C 00:01:00 2 95 °C 00:00:20 3 62 °C 00:00:30 4 68 °C 00:02:00 5 GO TO 2 REPEAT 30x 6 68 °C 00:05:00 7 4 °C HOLD
PCR on a DNA template
- One reaction mix contains:
- 10 µL of 5x Phusion HF Buffer
- 1 µL of dNTPs (10 mM each)
- 0,5 µL of Phusion High-Fidelity Polymerase
- Forward primer (10 pmol)
- Reverse primer (10 pmol)
- 1,5 µL of DMSO
- 1 µL of template
- DI water to 50 µL
- PCR program
# Temperature Time 1 98 °C 00:02:00 2 Ta 00:01:00 3 72 °C 00:01:00 4 98 °C 00:01:00 5 Ta 00:01:00 6 72 °C 00:01:00 7 GO TO 4 REPEAT 31x 8 98 °C 00:01:00 9 Ta 00:01:00 10 72 °C 00:06:00 11 4 °C HOLD
Restriction digest
A restriction digest is used to digest either vectors or inserts via restriction enzymes before ligation.
Protocol
- One reaction mix contains:
- DNA template (up to 3µg)
- 2 µL NEBuffer 4 (10x)
- 0,5 µL of restriction enzyme 1
- 0,5 µL of restriction enzyme 2
- 0,2 µL of 100x BSA (only when cut with SpeI-HF)
- DI water to 20 µL
- Incubate at 37° C for 1 hour
- Heat inactivate at 80° C for 25 minutes
