Team:Amsterdam/project/protocols/
From 2012.igem.org
Protocols
Transformation Protocol in DH5α (Invitrogen)
Notes:
- Do not shake the polypropylene tubes during the heat shock!
- To increase the yield, centrifuge gently (at low speed), remove the excess of medium and then spread on the plates.
- To determine transformation efficiency
Gel Electrophoresis
- 5 µl DNA ladder
- 5 µl sample + 5 µl H2O + 1 µl loading buffer
Preparation of LB Medium and Agar Plates
- Bacto Tryptone – 10 grams
- NaCl – 10 grams
- Yeast extract – 5 grams
Gibson Assembly
Preparation of reagents
- 3 ml of 1M Tris-HCl pH 7.5
- 150 ml of 2M MgCl2
- 60 ml of 100mM dNTP
- 300 ml of 1M DTT
- 1,5 g PEG-8000
- 300 ml of 100mM NAD
One-step isothermal DNA assembly protocol (Gibson Reaction)
- 8 µl 5X isothermal buffer
- 0.8 µl of 0.2 U.µl –1 or 1.0 U.µl –1 T5 exonuclease
- 4 µl of 40 U.µl –1 Taq DNA ligase
- 0.5 µl of 2 U.µl –1 Phusion DNA polymerase.
- 5 µl of DNA
- Water up to 40 µl
Notes:
- All isothermal assembly components can be stored at –20°C in a single mixture at 1.33X concentration for more than one year. The enzymes are still active after more than ten freeze-thaw cycles. The aliquots should be kept on ice until ready to use.
- The exonuclease amount is ideal for the assembly of DNA molecules with 20– 150 bp overlaps.
- Between 10 and 100 ng of each 6 kb DNA fragment was added.
- For larger DNA segments, proportional amounts of DNA should be added (for example, 250 ng of each 150 kb DNA segment).
DNA Precipitation
Notes:
- Keep the 100% ethanol at -20˚C. The ethanol must always be cold prior to use.
- Centrifugation at 4˚C is preferable.
Preparation of Competent E. coli
Preparation of Reagents
- 30 mM KOAc, 100 mM RbC12, 10 mM CaCl2, 50 mM MnC12, 15% glycerol, pH 5.8
- For 500 ml: 1.47 g KOAc (MW 98.14)
- 6.04 g RbC12 (MW 120.92)
- 0.74 g CaC12 (MW 147.02)
- 4.94 g MnC12-4H20 (MW 197.9)
- 75 ml glycerol
- pH to 5.8 with dilute acetic acid.
Protocol
Notes:
- Keep buffers, tips, tubes, rotors, etc. ice cold