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Revision as of 15:51, 29 October 2012 by Casey Durnan (talk | contribs) (Continuation of the Mutagenesis Reaction)
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Continuation of the Mutagenesis Reaction

  • On Tuesday, we prepared to combine our upstream and downstream fragments in the final mutagenesis step. First a high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. We then prepared our samples to amplify. Our approach was to mix the fragments together with and without primers to try to identify an effective combination to create our entire gene. For each set we also investigated the possible effect of template concentration and annealing temperature, for a total of 12 different combinations. Our nomenclature used is three-part, and outlined below.
  • 1.)Primer configuration
    • N = no primers used
    • P = outside primers used
  • 2.)Template DNA concentration
    • + = 1:20
    • - = 1:200
  • 3.)Annealing temperature for PCR, in °C
  • We used the same PCR protocol as last week.

  • On Thursday, we ran the gel for Tuesday's PCR products.
  • Our positive control was standard template DNA and associated primers from lab stock
  • Our negative control had outside primers and no template

  • We loaded our samples as follows:
  • Top Lane:
  1. 100 bp ladder
  2. P+55
  3. P+60
  4. P+65
  5. Positive Control
  6. P-55
  7. P-60
  8. P-65
  9. 100 bp ladder

  • Lower Lane:
  1. 100 bp ladder
  2. N+55
  3. N+60
  4. N+65
  5. Negative Control
  6. N-55
  7. N-60
  8. N-65
  9. 100 bp ladder

  • We ran these on a 1% agarose gel for 30min at 120V

  • Results:

  • Upper Lane

10.25.12 Primered Lanes.jpg

  • Lower Lane

10.25.12 Non-primered Lanes.jpg

  • Conclusions:
    • Our positive control checked ok, indicating working PCR reagents
    • Our negative control showed a faint band in the <100bp region, likely due to primers. Nothing else appeared, indicating an uncontaminated working stock.
    • Our non-primered set did not work. This should, in theory, so perhaps we need to adjust the thermocycle protocol.
    • Our primered set did amplify a numer of distinct bands. Our target length of ~400bp showed bands on both the upper and lower boundaries. One of these may be our intended product, as there is some range of error in marker ladders. Perhaps none of the bands are our product, however, and are just PCR artifacts.
    • Template dilution works well for either high or low concentrations. We will use 1:200 for further amplification.
    • Annealing temperature did not seem to vary results for this range.