Jeff:LabNotes/Microbiome/2011-2-7

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Post processing of Amplicons[edit]

Use amplicons extracted on 2-4-11

  • Run MDA using small amount of amplicons
  • Create following mix: 3 ul template and add 3 uL ALS buffer for 3 min
  • Neutralize with 3 uL NS buffer
  • Create master mix and add 16 uL to each reaction: 20.1 uL water, 15 uL 200 uM N6 primer, 7.5 uL 10x buffer, 2.4 uL dNTP, 3 uL enzyme
  • Run at 30 C for 2hrs followed by 85 C for 3 min
  • Purify with qiaquick column
  • All three amplicons were amplified: 1748 ug, 1388 ug, 2245 ug
  • A gel was run on these, but no band was seen around 12 kb. In fact, only the ladder was visible. I reran the gel 2 more times and still saw nothing.
  • Tried S1 nucleation and saw nothing on gel
  • Since I cannot confirm these amplicons, I need to start over