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USTC/Notebook/Top10 chem competent cell transformation
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'''[[Team:USTC/Notebook|< Back to Notebook]]''' | '''[[Team:USTC/Notebook|< Back to Notebook]]''' | ||
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| + | ===Regular Transformation protocol for Top10 cells=== | ||
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| + | Take out an appropriate aliquot of Top10 ChemComp cells from -80 freezer | ||
| + | Let cells thaw ON ICE! for ~5-10min. Cells MUST be kept cold until heat shock! This is critical! | ||
| + | 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. | ||
Revision as of 14:15, 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. Cells MUST be kept cold until heat shock! This is critical! 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.
