User:Anthony Salvagno/Notebook/Research/2009/10/29/Redoing Unzipping: Gel of Redone Anchor PCR

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Gel Results

SJK 12:37, 29 October 2009 (EDT)
12:37, 29 October 2009 (EDT)That is great that you have a foothold, now!  At least one successful reaction is so much easier to than all blank lanes.  Good work!  Can you use mfold to check the melting temperatures of the four oligos?  I had a dream last night that the pRL574 was so old that it had too many nicks to support PCR.  I guess that's not the case.  But something to keep in mind about very very old plasmid.
12:37, 29 October 2009 (EDT)
That is great that you have a foothold, now! At least one successful reaction is so much easier to than all blank lanes. Good work! Can you use mfold to check the melting temperatures of the four oligos? I had a dream last night that the pRL574 was so old that it had too many nicks to support PCR. I guess that's not the case. But something to keep in mind about very very old plasmid.

The image is blurry due to fog on the filter. I really should figure out some way to eliminate that. Anyways here is the lane readout:

  1. 1x Ladder
  2. 2x Ladder
  3. Tube 1
  4. Tube 2
  5. Tube 3
  6. Tube 4

See yesterday for contents of tube
It seems like Tube 1 almost worked and Tube 4 definitely worked. Tubes 2 and 3 definitely did not work. What happened in tube 1? Maybe extension is too long. I had this result happen to me before. I definitely think this is a thermal cycler thing now. There is relatively no difference in these reactions outside of the primers, and their locations are at most 100bp apart. I think the reaction worked depending on where I put them in the thermal cycler. I def need to take measurements. Anyways

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