Jeff:LabNotes/Microbiome/2010-8-30

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Revision as of 18:23, 3 December 2010 by >Jgole (Created page with ' == '''In well MDA using genomic DNA''' == ''Use MDA to compare results to pWGA'' *Prior to beginning, create NS and ALS buffers *ALS Buffer: 82 uL H20, 10 uL 1M DTT, 8 uL 5 M…')
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In well MDA using genomic DNA

Use MDA to compare results to pWGA

  • Prior to beginning, create NS and ALS buffers
  • ALS Buffer: 82 uL H20, 10 uL 1M DTT, 8 uL 5 M KOH
  • NS Buffer: 3 mL H2O, 3 mL 2M Tris, 4 mL 1N HCL
  • Make sure an even mix of the 2 buffers has a pH of 7-8 before moving on
  • Clean down hood with ethanol and UV tubes, arrays, gaskets, etc for 10 min
  • Use e coli gDNA with initial conc. 10^6 cell/uL
  • Create the following mix: 38uL .1% agarose, 12 uL gDNA, 6 uL C12 N6 labeled beads, 4 uL 3% BSA
  • Make another mix as an NTC (Use agarose instead of gDNA)
  • Add 3 uL of each mix to 2 separate squares and let settle for 5 min
  • Add 3 uL of ALS buffer to each and sit for 3 min
  • Create the following master mix while waiting: 15 uL 200 uM N6 primer, 7.5 uL 10x buffer, 17.1 uL H2O, 2.4 uL 25 nM dNTP, 3 uL Phi 29. This master mix is enough for 3 reactions
  • After 3 min, add 3 uL of NS buffer to each square to neutralize
  • Add 16 uL of master mix to each. Incubate at 30 C for 4 hr then 85 C for 3 min.