USTC/Notebook/Top10 chem competent cell transformation
From 2008.igem.org
(Difference between revisions)
Line 6: | Line 6: | ||
*Take out an appropriate aliquot of Top10 ChemComp cells from -80 freezer | *Take out an appropriate aliquot of Top10 ChemComp cells from -80 freezer | ||
- | *Let cells thaw | + | *Let cells thaw '''on ice!''' for ~5-10min until heat shock! |
*Aliquots are either 50µl (small PCR tubes) or 100µl (bigger tubes). | *Aliquots are either 50µl (small PCR tubes) or 100µl (bigger tubes). | ||
*Transform 50 μl of cells with 3-5µl of ligated DNA | *Transform 50 μl of cells with 3-5µl of ligated DNA | ||
- | *Keep | + | *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. | + | *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. | *Put cells back on ice for 5min. | ||
*Add your 50µl of cells to 200 μl LB media in a 1.5ml epp tube. | *Add your 50µl of cells to 200 μl LB media in a 1.5ml epp tube. |
Revision as of 14:18, 27 October 2008
Regular Transformation protocol for Top10 cells
- Take out an appropriate aliquot of Top10 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.