Difference between revisions of "Davidson Missouri W/colony PCR"
| Line 19: | Line 19: | ||
4. Conduct PCR according to the following thermal profile: | 4. Conduct PCR according to the following thermal profile: | ||
| − | *a. 94 C | + | *a. 94 C 5 minutes |
| − | *b. 30 cycles of: 94 C 15 seconds, 50 C 15 seconds, 74 C | + | *b. 30 cycles of: 94 C 15 seconds, 50 C 15 seconds, 74 C (60 seconds for each 1000 bp product size) |
| − | |||
5. Run 16 ul on polyacrylamide or agarose gel. | 5. Run 16 ul on polyacrylamide or agarose gel. | ||
6. Grow desired clones from reserved bacteria for use in plasmid preps. | 6. Grow desired clones from reserved bacteria for use in plasmid preps. | ||
Revision as of 15:34, 29 June 2011
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. Master Mix - Add 1 to the number of reactions and multiply this recipe by that total:
- 1 ul forward primer G0100 (10 pmol)
- 1 ul reverse primer G0101 (10 pmol)
- 7 ul dH2O
Dispense 9 ul Master Mix into each PCR tube
3. Add 10 ul 2X Promega GoTaq Hot Start Green Master Mix (http://www.promega.com/products/pcr/hot-start-pcr/gotaq-hot-start-polymerase/)to each reaction for 20 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 5 minutes
- b. 30 cycles of: 94 C 15 seconds, 50 C 15 seconds, 74 C (60 seconds for each 1000 bp product size)
5. Run 16 ul on polyacrylamide or agarose gel.
6. Grow desired clones from reserved bacteria for use in plasmid preps.
