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Team:Cambridge/Protocols/PCRcolony
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||Reverse Primer||2.5||0.5 µM | ||Reverse Primer||2.5||0.5 µM | ||
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| - | ||Template Cells||1.3 (from liquid culture or picked colony|| | + | ||Template Cells||1.3 (from liquid culture or picked colony)|| |
|- | |- | ||
||Taq polymerase 5u/µl||1||0.1 u/ µl | ||Taq polymerase 5u/µl||1||0.1 u/ µl | ||
Revision as of 18:10, 20 August 2012
Colony PCR:
Used to amplify DNA directly from cell culture, the reaction is the same as for normal PCR but with modified reaction composition and cycle settings, here shown for a 50 µl reaction using Taq polymerase.
| Reagent | Volume (µl) | Final Concentration |
| Water | 35.7 | |
| 10 mM dNTPs | 1 | 200 µM |
| 10 x NH4 buffer | 5 | 1x |
| Forward Primer | 2.5 | 0.5 µM |
| Reverse Primer | 2.5 | 0.5 µM |
| Template Cells | 1.3 (from liquid culture or picked colony) | |
| Taq polymerase 5u/µl | 1 | 0.1 u/ µl |
Please refer to the standard PCR protocol for the remainder of this protocol.
Notes on colony PCR
- This technique should only really be used for crude PCR assays, such as diagnostic PCR. If high quality DNA is desired, Miniprep followed by standard PCR should be used.
