840:153g:Projects/project22/2012/10/18

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PCR and Gel Electrophoresis Check

On Tuesday we decided that even though the gel electrophoresis was flawed due to anomalous specks on the gel...

Small gel 10.11.12.png <-- notice anomalous specks on gel

...we felt we had sufficient DNA to proceed to PCR. Our purpose was two-fold - to make sure the positive control worked (last time we ran PCR it did not) and to see if our DNA was truly present in the sample (PCR would not amplify the gene if DNA wasn't there).

On Thursday, we ran gel electrophoresis to check our PCR.

Small gel 10.18.12.png

As you can see, our positive control worked (4th lane from the left), but our samples (lanes 5 and 6 from the left) did not display the expected brightness. It is important to note that our annealing time was only 1 minute 30 seconds and our target gene is 1470 b.p., so that may have an effect on our outcome. It is also possible our DNA is compromised (and we will need to perform another DNA extraction) or our primers are flawed (aren't working properly).

For next Tuesday, we will increase the annealing time for PCR and see if we get better results. We will also grow up more cultures of S. marcescens and prepare for DNA extraction with alternate protocols obtained from Open WetWare (the last ones we used were obtained from the USDA).