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Team:Cambridge/Protocols/PCRcolony
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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. | 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. | ||
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||Reagent||Volume (µl)||Final Concentration | ||Reagent||Volume (µl)||Final Concentration | ||
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Revision as of 10:42, 13 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.
Safety considerations: It is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature and this may present a risk, depending on the PCR machine employed. For handling the cell culture, appropriate measures should be in place to deal with biohazardous waste.
