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Team:Cambridge/Protocols/Colony PCR
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|Water (reverse osmosis) || 35.7 μl || | |Water (reverse osmosis) || 35.7 μl || | ||
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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. | 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. | ||
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Latest revision as of 20:19, 21 September 2011
Contents |
Colony PCR
PCR can be used to amplify DNA directly from cell culture. We used this as a diagnostic tool to check that our constructs were successful. We used the standard PartsRegistry sequencing primers, [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00100 VF2] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00101 VR], and ran the PCR product on a gel to check the length.
Theory
Exactly the same as normal PCR except that the template consists of whole cells -- a colony picked from a plate or a small volume of liquid culture might be used -- and there is an initial heating step to lyse the cells.
Practice
Proceed as in normal PCR except with this modified reaction composition (for a 50μl reaction) and cycle settings:
| Name | 50 μl reaction | Final concentration |
|---|---|---|
| Water (reverse osmosis) | 35.7 μl | |
| 10mM dNTPs | 1 μl | 200 μM each |
| 10× NH4 buffer | 5 μl | 1× |
| Forward Primer 10 μM | 2.5 μl | 0.5 μM |
| Reverse Primer 10 μM | 2.5 μl | 0.5 μM |
| Template cells | 1.3 μl liquid culture or a picked colony | |
| Taq 5u/μl | 1 μl | 0.1 u/μl |
Settings of PCR machine:
| Step 1 (cell breakage) | 95°C | 6 min |
| Step 2 (cycle) | 98°C
55°C 72°C | 10 s
30 s 180 s |
| Step 3 (final extension) | 72°C | 5 min |
Safety
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.
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