Jeff:LabNotes/Microbiome/2011-12-6

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Revision as of 23:49, 9 December 2011 by >Jgole
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In Situ Tagmentatation

  • I believe that doing in situ tagmentation will help with contamination problem
  • The amplicons will be tagged prior to extraction, and if all the transposases are used up, there is no room for contamination in this step

Tagmentation

DNA in water  6.5 uL
HMW buffer  2 uL
1:10 diluted enzyme  2 uL
50x SYBR green .5 uL
----------------------
Total 10 uL
  • Pipette 10 uL mix onto array
  • 55C for 5 min
  • Look under microscope
  • The amplicon seems to be broken up, as represented by the dots as opposed to a continuous fluorescence signal
  • 2 amplicons were extracted due to evaporation issues


1st PCR round

DNA  6 uL
BST  1 uL
KAPA fast mix  15 uL
10 uM orange primer  .24 uL
10 uM blue primer  .24 uL
water  7.52 uL
--------------------------
Total  30 uL
  • 60 C 20 min, 72 C 3 min, 95 C 30s (95 C 10s, 58 C 30s, 72 C 2 min)x20, 72C 3 min

Results

  • The amplicons shot up meaning that tagmentation worked.
  • The gel looks funny. It does not seem that tagmentation worked due to the lack of a smear.

File:12-7-11 npcr.jpeg File:12-7-11 gel.jpeg