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Team:Cambridge/Protocols/PCRcolony
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Revision as of 15:00, 23 July 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 |
PCR machine settings:
| Temperature (oc) | Time (s) | ||
| Step 1 (cell breakage) | 95 | 360 | |
| Step 2 (Cycle 30x) | Denaturing | 98 | 10 |
| Annealing | 60 | 30 | |
| Elongation | 72 | 120 | |
| Step 3 (final extension) | 72 | 300 | |
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.
