Project:Sex typing with amelogenin/results-20120212

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To think about

  • DNA extraction: perhaps try several things, but especially:
    • Detergent
    • Salt
  • DNA extraction: try centrifuging DNA
  • DNA extraction: perhaps test by combining some extracted DNA with dilute EtBr solution.
  • Primers: can we tell that they are there? Try combining some with EtBr and viewing.
  • PCR: Do tests:
    • Without primers or template
    • Without template
    • Without master mix (?)
  • PCR: Follow Wikipedia's basic melt, anneal, extend temperatures and times.

Experiments to run:

Gel run 1

  1. PCR, no master mix, no template, just primers and 55 ul of buffer (Couldn't do this one, ran out of primers)
  2. PCR, no master mix, just template and primers and 5ul of buffer (Couldn't do this one, ran out of template and primers)
  3. PCR, master mix and primers, no template and 30 ul of buffer
  4. PCR, master mix, primers, and template (from top of tube)
  5. PCR, master mix, primers, and template (from bottom of tube)
  6. PCR, master mix (old one), primers, and template (from top of tube)
  7. PCR, master mix (old one), primers, and template (from bottom of tube)

DNA extraction

  • 6ml of saline solution in mouth
  • swish for 10 seconds
  • spit back into tube
  • centrifuge for 5 minutes
  • pour off supernatant (liquid on top) into sink.
  • suspend tube in boiling water for 10 minutes
  • place on ice for 1 minute
  • centrifuge for 30 seconds
  • transfer 200ul of supernatant to fresh tube. This contains DNA.

Protocol

  • 10 ul of primer (at a strength of 100 micromoles), x2 (so 10ul for each primer)
  • 25 ul of fast-start PCR Taq readymix
  • 30 ul of DNA.
  • 50 ul of mineral oil on top

PCR

  • D = 30 seconds at 98 degress
  • A = 40 seconds at 55 degrees (I think)
  • E = 60 seconds at 72 degrees
  • Repeat 25 times.
  • start: 14:22
  • end: 16:02

Gel lanes (viewed with the gel oriented so the lanes are at the top)

  1. Nothing
  2. PCR 3 above
  3. PCR 4 above
  4. PCR 5 above
  5. Ladder, 6ul
  6. PCR 6 above
  7. PCR 7 above
  8. Nothing

Started electrophoresis at 16:11 at 100v.

First visualisation taken after ~20 minutes of electrophoresis.

Second visualisation taken after ~45 minutes of electrophoresis.

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