Team:UCLA/Notebook/Spider Silk Genetics/21 May 2015

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5/21/2015

1. Test PCR with VF/R amd post-elution primers

• Test 20x and 23x cycles for VF/R and post-elution primers.
• Made new working 10 uM post-elution primers
• Set up 2x 25 uL reactions for each using same protocol as 5/19/2015 but with the following modifications.
  • Tested 23x and 20x cycles.

2. Results

• Cast a 2% TAE agarose gel to visualize results. Used NEB 50 bp ladder.

Fig. 1 Test amplification with biobrick sequencing primers (VF/R) and post-elution primers (P/E). The expected product for VF/R is 443 bp. The expected product for P/E is 170 bp.

• The post-elution primers were made from stock prior to this PCR, which means that the previous post-elution amplification was unsuccessful due to bad primers.
• The presence of a smear above the product bands is probably due to non-specific amplification. We should use fewer cycles. Along with results from 5/19/2015, which showed that 17 cycles was not quite complete, we will use 18 cycles for future amplifications.