Difference between revisions of "Davidson Missouri W/colony PCR"
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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. | 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. | + | 1. Determine the number of colonies to be tested. Plan to conduct PCR on control plasmids with and without the insert. |
| + | 2. Recipe for each reaction: ('''TIP: 1 uM = 1 pmol/uL''') | ||
| − | 2 | + | *20 pmol primer 1 (typically G00100) |
| + | *20 pmol primer 2 (typically G00101) | ||
| + | *12 uL 2X Promega GoTaq Hot Start Green Master ("Monster") Mix (http://www.promega.com/products/pcr/hot-start-pcr/gotaq-hot-start-polymerase/) | ||
| + | *Add dH2O for a '''total volume of 24 ul'''. | ||
| + | *'''If carrying out a large number of reactions, make a master mix of the water, 2X GoTaq, and primers in common between your samples.''' | ||
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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. | 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 | + | 3. Reserve bacteria from each PCR mixture by removing 1 ul and placing into 50 ul of media+appropriate antibiotic(s) in a labeled 1.5-mL tube. '''Vortex or agitate the tube, since the PCR mixture sinks to the bottom.''' Incubate the 1.5-mL tubes at 37 C. |
4. Conduct PCR according to the following thermal profile: | 4. Conduct PCR according to the following thermal profile: | ||
| − | *a. 94 C | + | *a. 94 C 10 minutes |
| − | *b. | + | *b. 20 cycles of: 94 C (15 seconds), 46 C (15 seconds), 74 C (60 seconds for each 1000 bp of product) |
| − | 5. Run 16 ul on polyacrylamide or agarose gel. | + | 5. Run 16 ul on a polyacrylamide or agarose gel. The distance between the primer binding sites and your part will add bases to your product size. |
| + | *Using G00100 (forward primer) adds 157 bp to parts in pSB1A2. | ||
| + | *G00100 adds 588 bp to pSB1A7 parts. | ||
| + | *G00100 adds 315 bp to pSB1C3 parts. | ||
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 17:12, 13 January 2012
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. 2. Recipe for each reaction: (TIP: 1 uM = 1 pmol/uL)
- 20 pmol primer 1 (typically G00100)
- 20 pmol primer 2 (typically G00101)
- 12 uL 2X Promega GoTaq Hot Start Green Master ("Monster") Mix (http://www.promega.com/products/pcr/hot-start-pcr/gotaq-hot-start-polymerase/)
- Add dH2O for a total volume of 24 ul.
- If carrying out a large number of reactions, make a master mix of the water, 2X GoTaq, and primers in common between your samples.
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 50 ul of media+appropriate antibiotic(s) in a labeled 1.5-mL tube. Vortex or agitate the tube, since the PCR mixture sinks to the bottom. Incubate the 1.5-mL tubes at 37 C.
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 (60 seconds for each 1000 bp of product)
5. Run 16 ul on a polyacrylamide or agarose gel. The distance between the primer binding sites and your part will add bases to your product size.
- Using G00100 (forward primer) adds 157 bp to parts in pSB1A2.
- G00100 adds 588 bp to pSB1A7 parts.
- G00100 adds 315 bp to pSB1C3 parts.
6. Grow desired clones from reserved bacteria for use in plasmid preps.
