User:Anthony Salvagno/Notebook/Research/2010/02/02/Jasper PCR Trial Try (6.1)

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Protocol

{{#widget:Google Spreadsheet |key=td-31iiRBYOjFr_YfRWS4zw |width=500 |height=300 }} I have changed the program to run at 55C and I used the NEB Taq buffer which has 1.5mM MgCl2 in it (or whatever I use I can't remember).

Results

This image stinks for several reasons: 1. Smeary gel (because of protein) 2. Didn't run straight. You can see this in the ladder alone. 3. Still hasn't run well. Maybe this is what happens? What does Cy3 or Cy5 do to DNA primer annealing. Maybe it somehow inhibits binding cause it is a huge protein. I just got new Taq (Platinum Taq from Invitrogen) and hopefully it is just a case of contamination and this will magically work. I believe in magic, don't you?

Try 6.2

{{#widget:Google Spreadsheet |key=tiBzGlDheFk3WNs2e1TMsIQ |width=500 |height=300 }} I increased extension time for this reaction to 1 min. And I added MgCl2 because of using a different PCR buffer. I have made the concentration 1.5mM.

Steve Koch 22:55, 2 February 2010 (EST): Definitely possible that labels can decrease the melting temperature, good thought. Also: is 1.5 mM sort of low?

Another idea: Can you quickly process the gel images: make them B/W, invert them (so that bands are dark on white background). THen post the original image and the processed to make it MUCH easier to see? Thanks!