Team:Cambridge/Protocols/Colony PCR


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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, VF2 and VR, and ran the PCR product on a gel to check the length.


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.


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
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



10 s

30 s

180 s

Step 3 (final extension) 72°C 5 min


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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