29 September 2014

Phenol-Chloroform and DNA

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DNA extraction for PacBio


Attempt 3 to get genomic DNA for PacBio sequencing from Aphaenogaster.

Modified the ant phenol-chloroform DNA extraction protocol combined with the Thermo Scientific Phenol/Chloroform Extraction and Ethanol Precipitation protocol.

  • Made STE buffer (0.1 M NaCl / 0.05 M Tris pH8 / 1mM EDTA)
  • for 100ml:
    • 10 ml 1M NaCl
    • 0.2 ml 0.5M EDTA
    • 84.8 ml H2O
  • Collected about 50 ants (weight = 0.27 g) from Molly Bog colony JSG002
  • Flash froze in liquid nitrogen
  • Crushed ants with mortar and pestle (bleach sterilized)
  • Mixed sample with STE + 0.5% SDS + 200ug/ml Proteinase K -for 10 ml:
    • 9.42 ml STE
    • 0.5 ml SDS 10%
    • 82.98 ul Proteinase K (24.1 mg/ml)
  • NOTE: the 10 ml digestino volume was way too much. I used this erroneously based on ‘DNA extraction for spermathecae’ protocol which listed ~3.8ml for 50 eggs. This measure was actually the volume to make, using only 50 ul per egg. I probably could have used only 1-2 ml of the mixture.
  • Incubated overnight at 55°C


Example of how to reproduce old R scripts: [http://blog.revolutionanalytics.com/2014/08/gran-and-switchr-cant-send-you-back-in-time-but-they-can-send-r-sort-of.html]

Are these better than packrat

  • packrat is for the future…stores packages at the time of your work
  • GRAN is more multi-purpose…allows you to roll-back to any previous version of R packages

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