Colony PCR Protocol
1. Pull out eight glycerol stock plates from the –80oC freezer and set on bench top to thaw. Be sure to remove the foil seal before leaving the plates to thaw.
2. Master mix:
| Per 100ul RXN | For one 96 well plate (100x) | For one PCR Run (8x 96-well plates) | |
| 10X PCR buffer w/ Mg2Cl | 10 ul | 1000 ul | 8000 ul |
| 10mM dNTPs | 2 ul | 200 ul | 1.600 ml |
| forward primer (10 pmol) | 1 ul | 100 ul | 800 ul |
| reverse primer (10 pmol) | 1 ul | 100 ul1 | 800 ul |
| ddH2O | 83.5 ul | 8.350 ml | 66.8 ml |
| template (50nmol) | 1 ul | 100 ul | 800 ul |
| Taq | 1.5 ul | 150 ul | 1.2 ml |
This is the template for a single 100 ul PCR reaction from plasmid. To adapt this protocol to bacterial colonies, just make up the RXN mix without the template added.
3. Run the appropriate programs on either the Biomek or the Multimek to aliquot 100 ul of PCR reaction (including TAQ) into Perkin-Elmer MicroAmp 96-well PCR reaction plates.
4. After aliquoting the reaction mix into the PCR plates, put the reaction plates on ice.
5. Using the 96-well pin inoculation tool, proceed to inoculate the PCR RXNs with the colonies from the appropriate plates.
6. Flame and ethanol dip between each inoculation. Treat the pin tool just like a sterile loop.
7. After inoculation, seal the plates with rubber gray mats and put them into the PE GeneAmp thermal cycler.
8. Run the appropriate program.
Clean the gray PCR mats byt rinsing them in 2% bleach and then water and then (optional) EtOH. Lay on bench top to dry or just pat it dry.
