Josh's Protocol
Sample Preparation
- Aliquot 50 uL sterile water into sterile PCR tubes, one tube per colony.
- Using a P20, scape off a colony and resuspend it in the water.
- Use 1 uL of this cell suspension as your PCR template
- Tubes can be stored at 4C for several days. Use 5 uL of cell suspension to inoculate a 5 mL overnight culture.
Reaction Mixture
- Per colony:
- 1 uL cell suspension
- 0.2 uL dNTPs
- 0.2 uL FWD and REV primers
- 1 uL 10x Taq Buffer (with MgCl2)
- 0.2 uL 1:10 Taq
- 7.2 uL water
- Note: Make a master mix of everything except the template. Aliquot 9 uL into a separate set of PCR tubes. Then transfer 1 uL of cell suspension to each tube. You can use a multichannel pipet to transfer up to eight templates at once.
- Run PCR as normal, giving 1 minute/kb for Taq.
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