"
USTC/Notebook/Top10 chem competent cell transformation
From 2008.igem.org
(Difference between revisions)
| Line 5: | Line 5: | ||
===Regular Transformation protocol for Top10 cells=== | ===Regular Transformation protocol for Top10 cells=== | ||
| - | 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 ON ICE! for ~5-10min. Cells MUST be kept cold until heat shock! This is critical! | + | *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). | + | *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 ON ICE 30min. This step is to let the salt from the ligation equilibrate over the cells. | + | *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. |
| - | 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. | + | *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. | + | *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 | + | *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. | + | *Plate 200 μl on the appropriate antibiotic plates. Use sterilized glass stick to spread. |
Revision as of 14:16, 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.
