840:153g:Projects/project4/2009/04/02

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Thursday 4/2: The Day of aprE Gene Amplification

Josh and Casy

  • The PCR products were ran on a gel for 30 minutes. The gel showed two bands of approximately 1000 base pairs, according to the ladder. This is the size of the aprE gene. However, the presence of the second band was not expected and the cause is unknown.
  • The gel was ran for another 30 minutes in order to separate the bands further, but they became blurred after running over the wells. The gel was discarded.
  • The concentration of the template DNA did not have a large impact on the results. The temperature, however, was best at 50°C.
  • The PCR success may be attributed to one of two factors. This is the first PCR using DNA from B. subtilis 168. Also, the primers used in this PCR were mutated to prevent hairpin and dimer formation.
  • Another PCR was setup for tuesday. This was setup with 0.1μL of DNA and at 50°C.
  • Tuesday, the PCR product will be digested, ran on a gel, and the fragment(s) will be isolated and extracted from the gel.

Derek and Katy

  • We did a PCR amplification of wintergreen using three temperatures, two concentrations, and two primers so that we could find the best combination to enhance amplification.
  • We used 48, 52, and 56 degrees for the temperatures.
  • We used DNA primers 7 and 8 because they were the ones that showed up on our last gel.
  • We used concentrations 1.0mL and .1mL to see if there was too much DNA in our previous PCR's.
  • By doing this we hope to find combinations that yield better results than others.

Oggie

  • Performed several tests using PCR in hopes of troubleshooting and determining the cause of the nprE PCR failures
  • All results looked the same regardless of the forward primer differences
  • Tests involved samples containing just the reverse primer, and no primer at all
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