"
Team:Cambridge/Protocols/PCRcolony
From 2012.igem.org
(Difference between revisions)
(→Colony PCR:) |
(→Colony PCR:) |
||
| Line 52: | Line 52: | ||
*This technique should only really be used for crude PCR assays, such as diagnostic PCR. If high quality DNA is desired, [[Team:Cambridge/Protocols/MiniPrep|Miniprep]] followed by [[Team:Cambridge/Protocols/PCRProtocol|standard PCR]] should be used. | *This technique should only really be used for crude PCR assays, such as diagnostic PCR. If high quality DNA is desired, [[Team:Cambridge/Protocols/MiniPrep|Miniprep]] followed by [[Team:Cambridge/Protocols/PCRProtocol|standard PCR]] should be used. | ||
*If taking a colony from a plate, resuspend the cells in ~50μl of water. Take 1 μl of this resuspension and use it as your template. Failing to do so will result in you having too much genomic DNA in your reaction, which will interfere with gel electrophoresis - you can tell if this has happened if a bright band appears where your wells are on the gel. | *If taking a colony from a plate, resuspend the cells in ~50μl of water. Take 1 μl of this resuspension and use it as your template. Failing to do so will result in you having too much genomic DNA in your reaction, which will interfere with gel electrophoresis - you can tell if this has happened if a bright band appears where your wells are on the gel. | ||
| + | *Resuspended cells can be stored in the fridge for a couple of days for eventual plating, if colony PCR proves successful. | ||
Revision as of 13:59, 26 September 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.
- If taking a colony from a plate, resuspend the cells in ~50μl of water. Take 1 μl of this resuspension and use it as your template. Failing to do so will result in you having too much genomic DNA in your reaction, which will interfere with gel electrophoresis - you can tell if this has happened if a bright band appears where your wells are on the gel.
- Resuspended cells can be stored in the fridge for a couple of days for eventual plating, if colony PCR proves successful.
