After receiving the iGEM Distribution Kit, and transforming our
laboratory chemically competent cells with provided plasmid.
We discovered that our plates contain an abnormally large number
of colonies. By double checking the supplemented iGEM documentation,
we could not find any information explaining the issue. However,
after checking the literature, we realised that the used E. Coli
strain was XL 10 Gold, which is already resistant to Chloramphenicol
(the same resistance as given by iGEM kit plasmids).
In order to help other teams not to repeat our mistake, we troubleshooted iGEM kit documentation and studied the literature to write about most commonly used strains of E. Coli, their antibiotic resistance and primary use, and provided general guidelines.
|Strain name||Natural rasistence||Primary Use|
|DH10B||Streptomycin||MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph.|
|DH5alpha||General cloning and storage of common plasmids, blue/white screening.|
|JM109||General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient; good strain for cloning repetitive DNA.|
|MC1061||Streptomycin||Parent of DH10B/TOP10 and derived strains, common lab cloning and storage strain.|
|NEB Stable||For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences with the potential to recombine.|
|Stbl2||JM109-derived. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences with the potential to recombine.|
|Stbl3||Streptomycin||Derived from HB101. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences with the potential to recombine. This strain is endA+, so column-based DNA preps require an additional wash step.|
|TOP10||Streptomycin||MC1061 derivative. General cloning and storage, blue/white screening.|
|XL10 Gold||Tetracycline and Chloramphenicol||High competency cloning and propagation of large plasmids, ligated DNA, and libraries, nalidixic acid resistant.|
- Thaw on ice one tube (approximately 200 ul) of chemically competent cells. Place 1.5 ml microcentrifuge tubes on wet ice.
- Gently mix cells with the pipette tip and aliquot 50 μl of cells for each transformation into a 1.5 ml microcentrifuge tube.
- Add 1 to 5 μl (1-10 ng) of DNA to the cells and mix gently. Do not mix by pipetting up and down.
- Incubate tubes on ice for 30 minutes.
- Heat shock cells for 20 seconds in a 42°C water bath without shaking.
- Place tubes on ice for 2 minutes.
- Add 950 μl of pre-warmed medium of choice to each tube.
- Incubate tubes at 37°C for 1 hour at 225 rpm.
- Spread 20 μl to 200 μl from each transformation on pre-warmed selective plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, plate 100 μl on an LB plate containing 100 μg/ml ampicillin.
- Store the remaining transformation reaction at +4°C. Additional cells may be plated out the next day, if desired.
- Incubate plates overnight at 37 °C.
Follow the manufacturer's recommendations for making LB plates, be sure to add the agar, not agarose. After autoclaving, and when the agar has cooled enough that it's not too hot to touch (to approximately 50°C, about 1 to 1.5 hrs waiting), add antibiotics as follows:
- Ampicillin – add 1 ml ampicillin (at 100 mg/ml) per litre of agar to obtain a final concentration of 100 ug/ml.
- Kanamycin – add 1 ml kanamycin stock (at 50 mg/ml) per litre of agar to obtain a final concentration of 50 ug/ml.
- Tetracycline – add 1 ml tetracycline stock (at 15 mg/ml) per litre of agar to obtain a final concentration of 15 ug/ml.
- Chloramphenicol – add 1ml chloramphenicol stock (at 25mg/ml) per litre of agar to obtain a final concentration of 100ug/ml.