Difference between revisions of "E. coli transformation"
| Line 1: | Line 1: | ||
| − | Using the puch tool, puch out the appropriate DNA. | + | Using the puch tool, puch out the appropriate DNA. Clean the tool between punches. |
| + | |||
| + | Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice. | ||
Add 2µL of DNA in TE to 50µL of competent cells (TOP10) | Add 2µL of DNA in TE to 50µL of competent cells (TOP10) | ||
| Line 9: | Line 11: | ||
Recover on ice for 5 min. | Recover on ice for 5 min. | ||
| − | Add 200 µL SOC media | + | Add 200 µL SOC media. |
Incubate at 37ºC for 2 hr while the tubes are rotating. | Incubate at 37ºC for 2 hr while the tubes are rotating. | ||
| + | |||
| + | Plate 250µL on an LB plate with the appropriate antibiotic. | ||
Revision as of 12:58, 21 March 2008
Using the puch tool, puch out the appropriate DNA. Clean the tool between punches.
Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.
Add 2µL of DNA in TE to 50µL of competent cells (TOP10)
Allow the DNA and competent cells to sit on ice for 20 minutes
Heat shock at 42ºC for 60 sec in water bath.
Recover on ice for 5 min.
Add 200 µL SOC media.
Incubate at 37ºC for 2 hr while the tubes are rotating.
Plate 250µL on an LB plate with the appropriate antibiotic.