(modified from Protocol by Todd Eckdahl, Missouri Western State University)
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:
Per Reaction (you might want to make a cocktail, rather than multiple individual reactions)
1 µL forward primer (20 pmol = 0.2 µL of 100 µM oligo stock solution)
1 µL reverse primer (20 pmol = 0.2 µL of 100 µM oligo stock solution)
10 µL dH2O
12 µL 2X Monster Mix (Green solution from Promega)
24 µL total volume
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 and put in 37° C incubator.
4) Conduct PCR according to the following thermal profile:
94° C 10 minutes
20 cycles of:
94° C 15 seconds
46° C 15 seconds
72° C 30 seconds (time varies depending on the size of insert; rule of thumb is 1 minute per kb of DNA being amplified)
Hold at RT°
5) Run reaction on appropriate percentage agarose gel. If there is no insert, then the PCR product will be 258 bp in size.
6) Add 1.9 mL of media to desired clones from reserved bacteria (step 3 above) for use in plasmid preps. Do your normal MiniPrep procedure.
© Copyright 2007 Department of Biology, Davidson College, Davidson, NC 28036
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