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Regular Transformation protocol for Top10/DH5α cells
- Take out an appropriate aliquot of Top10/DH5α ChemComp cells from -80 freezer
- Let cells thaw on ice! for ~5-10min until heat shock!
- Aliquots are either 50µl (small PCR tubes) or 100µl (bigger tubes).
- Transform 50 μl of cells with 3-5µl of ligated DNA
- Keep on ice 30min. This step is to let the salt from the ligation equilibrate over the cells.
- Heat shock 90 sec at 42C. You can set a thermocycler to 42*C instead of making a water bath.
- Put cells back on ice for 5min.
- Add your 50µl of cells to 200 μl LB media in a 1.5ml epp tube.
- Incubate at 37 C for 1 hour. You can just place the 1.5ml epp tubes in a little plastic cup and put them on the *shaker in the 37*C room.
- Using 1.5ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
- Ampicillin and kanamycin appear to do fine with 1 hour growth.
- Plate 200 μl on the appropriate antibiotic plates. Use sterilized glass stick to spread.
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