User:Tara K. Luckau/Notebook/Team ConGen/2010/11/08

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Contents

Gel Scun2 F-R with DNA Gradient

Cast Gel

  • 270µL 1x TAE + 5.4g agarose
  • hot plate + stir bar
  • 27µL Gel Red (added when agarose turned clear but still hot, used stir bar)

Load Gel

  • 5µL NanoPure H2O + 5µL sample
    • (to conserve loading dye)
    • (don't need loading dye in every sample since it's in ladder)
  • 10µL ladder / loading dye mix

Run Gel

  • 130C, 2 hrs

Image Gel

  • UGH!
  • 3 faint bands

Results

  • Very faint bands at E8, E10, E12, corresponding to DNA concentration of 1x, Buffer G, Temperatures 61, 63, 65
  • around 150 bp band

Next Steps

  • there's obviously a problem:
    • reagent sucks (made wrong, not stored properly, expired)
    • inhibition
  • Start weeding out the problem
    • check math on dNTPs, ladder (too bright?)
    • try different Taq?
    • try Scun22 primers



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