User:Brian P. Josey/Notebook/2009/12/07

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Gel Continuation

I am setting up the second gel from the double stranded PCR from last Thursday. Today I am running the B Samples, and as before I am running the six samples with a ladder as a control. As usual, each lane has a volume of 15 μL of the sample and 3 μL of the 6X dye, giving a final volume of 18 μL per lane. Going left to right, the lanes are:

  1. Ladder
  2. 1B: pRL574-F853-dig/pRL574-R5263-bio
  3. 2B: pRL574-F853-dig/pRL574-R2044-bio
  4. 3B: pRL574-F853-dig/pRL574-R2008-bio
  5. 4B: pRL574-F834-dig/pRL574-R5263-bio
  6. 5B: pRL574-F834-dig/pRL574-R2044-bio
  7. 6B: pRL574-F834-dig/pRL574-R2008-bio

PCR 12-3-09 B.JPG

As you can see lanes 3, 4, 6, and 7 all developed nicely, while lanes 2 and 5 failed to develop the same length of DNA. This indicates that the 2B, 3B, 5B, and 6B samples were all successful, and the 1B and 4B samples were not. As I noted on Friday, where a similar success pattern developed, I had guessed that the pRL574-R5263-bio primer was not functioning, and this seams to confirm this. In both the A and B samples, all the lanes that featured the pRL574-R5263-bio failed to develop even slightly.

Also there isn't nearly as much smearing in this gel as there was in the one from Friday, which is a good thing. Koch suggested that the smearing could have been the result from running the gel too long, improper magnesium concentration, or something else. Of course, the concentration for these samples is about half of what the concentration for the A samples was, but I between two gels I can't tell if that is the cause of the smearing. As for the run time of the gel, I ran this one for about five minutes shorter to be on the safe side. This gel was ran for about seven minutes, while the other one was around twelve minutes. Whether it was the shorter run time, the lower concentration of magnesium, or another factor, this gel is much better than the other one, and it is clear that we have four successful samples from each batch, for a total of eight, and that all the samples that had the pRL574-R5263-bio primer in it were not as successful.