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Team:Waterloo/Notebook/Protocols/Colony PCR
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<b><u><big>Colony PCR </big></u></b><br> | <b><u><big>Colony PCR </big></u></b><br> | ||
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8. Add 20 µL of Master Mix to each PCR tube.<br> | 8. Add 20 µL of Master Mix to each PCR tube.<br> | ||
9. Centrifuge briefly in the PCR machine. Use the pulse option.<br> | 9. Centrifuge briefly in the PCR machine. Use the pulse option.<br> | ||
| - | 10. | + | 10. Start the PCR machine on the desired cycle.<br> |
Latest revision as of 22:51, 28 October 2008
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Colony PCR
Materials
- 20 µL of Master Mix
Calculations for Master Mix
___ number of colonies (and control)
(___+1) x 0.5µL dNTPs
(___+1) x 15µL PCR H2O MQ
(___+1) x 2.5µL Taq Buffer
(___+1) x 0.5µL fwd primer
(___+1) x 0.5µL rev primer
Instructions
1. Prepare agar plate with the correct antibiotic. Draw a grid with as many squares as the colonies you’re screening.
2. Label PCR tubes for each colony and one control (no DNA or colony)
3. Add 6µL of PCR H2O MQ to each PCR tube
4. Pick a colony from the agar plate with a pipette tip and resuspend in 6 µL PCR H2O. (if inoculating LB, resuspend in 7 µL PCR H2O)
5. Add 1 µL of resuspended DNA to a grid square. Incubate overnight at 37C.
6. Optional: Add 1 µL of resuspended DNA in corresponding LB tube and inoculate overnight
7. Prepare master mix. Vortex master mix before adding Taq. Once you added Taq mix gently.
8. Add 20 µL of Master Mix to each PCR tube.
9. Centrifuge briefly in the PCR machine. Use the pulse option.
10. Start the PCR machine on the desired cycle.
