User:Tara K. Luckau/Notebook/Team ConGen/2011/03/11

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Scun2 FAM Gradient PCR

  • Moving on with Scun2:
  • use labelled forward primer (Scun2 F FAM) (get ready to genotype)
  • full temperature and MgCl2 gradient to find annealing temperature (no touchdown)
  • 2011 03 11 PCR.jpg

Scun2 FAM Gradient Gel

  • Pour: 270mL 1x TAE + 5.4g agarose + 27µL GelRed
  • Load: 1µL gel load buffer + 5µL PCR product
  • Run: 1 hour, 150V
  • 20110311 GelFull.jpg

  • Zoom in on the only part that had amplicon:
  • 20110311 GelZoom.jpg
  • Amplicon at ~150bp consistent with expected amplicon size - nice and clean
  • amplicon present across many temperatures, but more so on lower range (55-60°C)

  • try: Buffer G, 58°C, 15µL reaction on diversity panel to send to frag