Cfrench:PfuPCR
2. Cloning DNA for new biobricks using Pfu polymerase
Pfu is a proof-reading polymerase from Promega. They claim that it is the most accurate polymerase available for PCR, making far fewer errors than Taq. Unlike most other archaeal polymerases, it uses a protocol almost identical to that of Taq, except that a longer extension time is required. I now recommend that Pfu rather than Taq should be used for all primary PCR cloning, and Taq should be used only for confirming the identity of clones (described later). Taq is cheaper, faster and usually works OK for cloning, but just every now and then it throws up an unexpected mutation which can waste a lot of time.
Template: for primary cloning, I recommend using a cell suspension. To prepare a cell suspension, place 200 microlitres of sterile water in a 1.5 ml microcentrifuge tube, add a generous loopful of cells from a plate, and resuspend by pipetting up and down with a P200. Alternatively you can spin some cells down from liquid culture (0.2 to 1.0 ml depending on density), remove the supernatant (to autoclave waste) and resuspend the cells in 200 microlitres of sterile water.
You can also use one microlitre of a genomic DNA prep as template, but in my experience it does not usually give better results than just using cells.
Reaction mixture
- Water: 39.6 μl
- 10 x reaction buffer: 5 μl
- forward primer: 1 μl
- reverse primer: 1 μl
- dNTP mix (10 mM each): 1 μl
- cell suspension: 2 μl
- Pfu polymerase: 0.4 μl
______________________________
- Total Volume: 50 μl
PCR protocol
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: 2 minutes/kb at 72˚C
Chase: 10 minutes at 72˚C
Cool to 7˚C and hold.
Notes: the Progene thermal cycler is set to run this protocol as programs 2 (denature), 3 (cycle), 4 (chase) and 5 (cool). You will need to alter the annealing temperature and extension time to suit the primers and expected product size.
Analysis
Generally: in a 1.5 ml microcentrifuge tube, mix 5 microlitres of PCR product with 5 microlitres of water, add 2.5 microlitres of loading buffer, mix, spin for a few seconds in a microcentrifuge to get the liquid to the bottom of the tube, and run the entire mixture in one lane of an agarose gel with suitable size markers.
