User:Tara K. Luckau/Notebook/Team ConGen/2011/01/12

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Revision as of 16:26, 12 January 2011 by Tara K. Luckau (talk | contribs) (Gel Timbers (by Tara))
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PCR Timbers (by Tara)

  • PCROpt Timbers.jpg

Extract Sceloporus undulatus

  • continued from 11 January 2011
  1. add 1.5μL RNAse
  2. incubate 30 min in 37°C water bath
  3. allow to cool to room temperature
  4. add 100µL Protein Precipitation Solution; vortex; centrifuge 10 min to pellet
  5. transfer supernatant (contains DNA) into new tube
  6. add 300µL 100% isopropanol; invert; centrifuge 10 min to pellet
  7. pour off supernatant
  8. add 300µL 70% ethanol; invert; centrifuge 10 min to pellet
  9. pour off supernatant; let air-dry
  10. rehydrate with 50-200µL Tris-Cl pH 8.4 (10mM); incubate overnight at room temperature

Gel Timbers (by Tara)

  • File:20110112 TimberTKL.tif
  • bands! and they seem to have the same pattern as yesterday's PCR by Rulon (NH 24 has fainter larger band, and smeary smaller band)
  • no big difference between Rulon's or Tara's dNTPs
  • no big difference between Rulon's or Tara's Buffer and MgCl2
  • conclusion: dNTPs and Buffers are not cause of PCR problems!