This procedure uses PCR to determine the size of DNA cloned into a plasmid from a single colony on a transformation plate, while reserving some of the bacteria for further growth and plasmid preparation.
1. Determine the number of colonies to be tested. Plan to conduct PCR on control plasmids with and without the insert. Assemble the following PCR mixture:
- 2 ul 10X reaction buffer
- 2 ul 2 mM dNTPs (working concentration 200 uM each)
- 1 ul forward primer G0100 (20 pmol)
- 1 ul reverse primer G0100 (20 pmol)
- 1 ul Taq DNA polymerase (2.5 u)
- 12 ul dH2O
- 19 ul total
2. Use a micropipette tip to pick a single putative colony off a plate. Insert the tip into the PCR mixture and pipette up and down.
3. Reserve bacteria from each PCR mixture by removing 1 ul and placing into 100 ul of LB + Amp in a labeled tube.
4. Conduct PCR according to the following thermal profile:
- a. 94 C 10 minutes
- b. 20 cycles of: 94 C 15 seconds, 46 C 15 seconds, 74 C 30 seconds
- c. 74 C 5 minutes
5. Add 5 ul 5X loading buffer and run 14 ul on polyacrylamide or agarose gel.
6. Grow desired clones from reserved bacteria for use in plasmid preps.