User:Bryan Barney/Notebook/Cancer magister PCR/2011/01/10

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Template DNA description

  • Experiment was to check the template DNA (both 1/5 dilutions and neat) for possible degradation.
  • 1/5 dilutions were stored at 4C for ~1 month prior to testing.
  • Neat stored at -20C for 1 year.

DNA concentrations for the samples is unknown

PCR Conditions

PCR performed as standard (I will add below at a later date)

  • samples:
    • WANQ01 through WANQ08 (1/5 dilutions)
    • WANQ01 through WANQ08 (neat)

Gel electrophoresis

2% agarose gel run
50V for 5 minutes
100V for 40 minutes

Gel picture

Gel Image

Top Row:

Lane 1 = 3μL custom 100bp ladder
Lane 2 = (-) control
Lane 3 = WANQ01 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 4 = WANQ02 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 5 = WANQ03 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 6 = WANQ04 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 7 = WANQ05 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 8 = WANQ06 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 9 = WANQ07 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 10 = WANQ08 1/5 dilution (3μL template, 7μL TBE buffer)
Lane 11 = 2μL custom 100bp ladder

Bottom Row:

Lane 1 = 3μL custom 100bp ladder
Lane 2 = (-) control
Lane 3 = WANQ01 neat (3μL template, 7μL TBE buffer)
Lane 4 = WANQ02 neat (3μL template, 7μL TBE buffer)
Lane 5 = WANQ03 neat (3μL template, 7μL TBE buffer)
Lane 6 = WANQ04 neat (3μL template, 7μL TBE buffer)
Lane 7 = WANQ05 neat (3μL template, 7μL TBE buffer)
Lane 8 = WANQ06 neat (3μL template, 7μL TBE buffer)
Lane 9 = WANQ07 neat (3μL template, 7μL TBE buffer)
Lane 10 = WANQ08 neat (3μL template, 7μL TBE buffer)
Lane 11 = 2μL custom 100bp ladder

Conclusions

PCR mastermix works - all components are OK for amplification

  • Very little amplification from either of the sample sets, but there is positive amplification in the right size range (Cma117 should be ~300bp, see bottom row lanes 5, 7, and 9)
  • slight amplification in other lanes
  • sporadic amplification could be due one of the following:
  1. no template DNA in the original vials
  2. a PCR inhibitor present in the neat vials and the dilutions have degraded
  3. null alleles in the sample set (unlikely at this high frequency)