Quick ligations using Roche Rapid DNA Ligation Kit



Transformation of JM109 competetent cells (Promega, cat# L2001, >108 CFU/µg))

1. Take out frozen competent JM109 cells from the –80 ˚C freezer and put right away on ice. Let them thaw slowly on ice.

2. When thawed, add half the ligation mix (10 µl) to 50 µl of competetent cells on ice (use an eppendorf tube); incubate one hour on ice. (the remaining competent cells can be refrozen at –80 ˚C; however mark the tube on top with a dot to indicate that these are no longer highly competent cells, but still good for moving DNA around)

3. Heat shock the transformation mix (competent cells) at 42 ˚C for 45 seconds then put on ice for a couple of min.

4. Add 500 µl of LB and incubate in warm room (37 ˚C) with shaking for 1 hour.

5. Spin down cells and resuspend in 100  µl LB. Plate everything on an LB-Amp plate and incubate plate overnight in warm room. Pick single colonies and screen.

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