User:Anthony Salvagno/Notebook/Research/2011/02/07/PCR results and Thoughts

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PCR Results

I'll have to figure out a way to get a good picture up, but the result of the gel is a smeary product with at least 3 distinct bands hidden among the giant smear. Meaning that we got successful PCR but a failed product. At least changing the temperature did something. Now I have to figure out how to fix it. I'll do some reading and see what I can dig up.

Thoughts

Possible Solutions:

  • Raise the annealing temp a couple of degrees. Changing this could result in more specific binding instead of anywhere.
  • Check the sequence for other possible binding sites, I don't know how to go about this though
  • Run a check reaction from Molecular Cloning
    • There is a protocol that describes how to run a reaction with controls and other variables so you can determine what goes wrong in the event of a messy reaction. I should probably look into this first before trying to guess at what is wrong.

According to MC, I should decrease the annealing time or increase the annealing temp. I could also try reducing the amount of template DNA. It would seem I'm getting some nonspecific primer binding and possible amplification of primer-dimers (due to the smearing). How do I know if the time needs to be decreased or if the temp should be increased?

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