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August 3rd, 2011

  • Reduced the number of oligos in each PCA reaction to 3 groups of 8 (19-26; 27-34; 35-42) and 2 groups of 6 oligos (43-48; 49-54). PCR purified the products and loaded the DNA in the gel. We encountered an issue that occurred a few time in the past, however, where the DNA would float up out of the wells instead of staying at the bottom of the well. We finally realized that this was because warm water floats to the top, which happens because we elute DNA from the spin column with water at 55C. After putting the DNA into the freezer for a couple of minutes, we reloaded the samples, which worked perfectly. Gel analysis showed that we had very little DNA at the lengths we wanted:

1) ladder

2) PCA oligos 19-26 (PCR purified)

3) PCA oligos 27-34 (PCR purified)

4) PCA oligos 35-42 (PCR purified)

5) PCA oligos 43-48 (PCR purified)

6) PCA oligos 49-54 (PCR purified)

  • This may be a result of known issues dealing with the assembly of GFP (Dr. Kwon). We decided that we should go ahead and try to assemble VEGF with some modifications. Instead of trying to assemble a 2kp DNA segment, we would assemble VEGF with some restriction enzyme sites so we could biobrick or digest and ligate our desired construct into the plasmid. Thus, we developed and optimized the VEGF sequence.