User:Anthony Salvagno/Notebook/Research/2009/04/16/PCR Reaction

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PCR Setup

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  1. That is the prep for a master mix. From there aliquot 90uL to 5 PCR tubes on ice. We don't want the Taq Polymerase to start before it gets to the PCR machine.
  2. Run "Anchor" Program. The settings are as follows:
    1. 94C for 2min (Cycle starts after this step)
    2. 94C for 30s
    3. 60C for 1min
    4. 72C for 2min (End Cycle)
    5. Set to run for 35 cycles
    6. 72C for 9min
    7. 10C for 99hours (basically for an overnight).

Reaction Participants Prep

Not all solutions were the way I needed. So there is some prep work:

  • pRL574 is 100x of what I need
  • F834 is ~7x (6.9x)
  • R1985 is ~10x (9.7x)
    • Steve Koch 00:45, 17 April 2009 (EDT): It's absolutely critical that you use the correct primers here. You want the pRL574-F834 primer to be the DIG version. And you do NOT want biotin on the reverse primer. We should have 100 micromolar stock of these primers, either in Osley's freezer, or in ours. You can easily make your own dilutions from these, and you may want to do that if you're at all uncertain of the identify of the dilutions you have now.

How PCR Works

I just learned this today so here goes.

  • Ramalldf 03:08, 24 April 2009 (EDT):Wow, your notebook is looking real good Ant :) One thing to look out for when doing PCR is that the lid is on tight and that it is heating. We have an older PCR machine in our lab right now and we have to be very careful because the lid doesn't automatically turn on (once the thermocycler reaches 94 degrees, all of your sample will condense at the top of the tube and this may affect the effectiveness of your reaction, and total volume of working rxn as well). From what I remember though Osley's PCR machines are both automatic so this should be less of a problem for you.
  • One other thing that the thermocycler is very useful for is a replacement for a heating/cooling bath. It is MUCH easier to do this than to try to adjust the temperature on one of those stupid heat blocks or to keep microwaving a water bath until you reach the right temperature.