Jeff:LabNotes/Microbiome/2011-12-6
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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.