7. Screening colonies by PCR
This is an alternative to plasmid DNA minipreps, described in section 8. For screening purposes, where there is no intention to clone the PCR product, Taq should be used rather than Pfu, as it is cheaper and faster and does not eat primers. The following protocol assumes that GoTaq polymerase from Promega will be used. If a different preparation (or more specifically a different buffer) is used, the reaction mixture will need to be modified – check the instructions that come with the polymerase and buffer. Especially note that some buffers include magnesium chloride, whereas Promega Gotaq comes with a separate tube of 25 mM magnesium chloride. For any variety of Taq (as opposed to an archaeal proof-reading polymerase such as Pfu, Pfx, Tth, Vent etc.) the cycling protocol should be fine.
To screen 6 colonies or pools:
- 1. For each colony or patch, prepare a cell suspension as follows. Place 200 microlitres of sterile water in a 1.5 ml microcentrifuge tube. Add a generous loopful of cells. (It may be difficult to get enough cells from small colonies, so it may be necessary to patch the cells to a fresh plate and grow them for a few hours or overnight). Suspend the cells by pipetting up and down. If you have a large number of colonies to screen and a low expectation of success, you can pool clones for the initial screening step, and if any pool comes up positive, do a second screen to determine which clone was positive.
- 2. Prepare reaction mixture in a 1.5 ml microcentrifuge tube as follows:
- water: 86.8 microlitres
- 5 x buffer: 28 microlitres
- 25 mM MgCl2: 8.4 microlitres
- primer 1: 2.8 microlitres
- primer 2: 2.8 microlitres
- dNTP mix, 10 mM each: 2.8 microlitres
- Taq polymerase: 1.4 microlitres
- Total volume: 133 microlitres
- Mix the solution by pipetting, and immediately dispense 19 microlitres into each of 6 PCR tubes (0.5 ml). To each tube add 1 microlitre of cell suspension. Place in the PCR machine and start the amplification as quickly as possible.
Alert readers will notice that the reaction mixture is enough for 7 tubes rather than 6. In my experience, if you make up exactly enough for 6 tubes there is never quite enough, so I usually make up enough for 0.5 or 1 extra tube.
Note: For historical reasons, I usually use primer working solutions as 50 pmol/microlitre, but some other people use 10 pmol/microlitre, which seems to work at least as well, so I may change this in future. Feel free to try it if you like.
- Denature: 2 minutes at 95˚C
- Cycle: 30 x denature: 30 seconds at 95˚C; anneal: 30 seconds at 55 to 58˚C depending on primers; extend: 1 minute/kb at 72˚C
- Chase: 10 minutes at 72˚C
- Cool to 7˚C and hold.
Note that Taq is faster than Pfu, so the extension time is 1 minute per kb rather than 2 minutes per kb.
When the reaction is complete, mix 5 microlitres of PCR reaction with 5 microlitres of water and 2.5 microlitres of loading buffer, and run in one lane of an agarose gel with appropriate molecular weight markers.