Rui:LabNotes/Haplotyping/2011-8-17

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Testing for haplotyping method[edit]

  • First test: protease treatment after tagmentation
I once tested it as Athurva used (1ul, 1:100 dilution) , however it showed delayed amplification.
This time, I reduced the protease (0.4ul and 0.2ul vs 1ul), as  I used less transposon (0.4, 1:50d) than others (1ul, 1:50d).
  • Second test: 2nd round of PCR with beads or completely purified DNA

Procedure[edit]

  • cells aliquot on 8/8/11 [1]
  • 1 ul of 10 cells/ul + 30ul lysis buffer (1X, made on 8/3/11 [2]); 37C, 30min
  • Add 0.3ul protease to tube, immediately aliquot 1ul/tube for 20 tubes; 50C, 15min, 70C, 15min
  • Tagmentation with 0.4ul of 1:50d transposon
  • Protease treatment: none, 0.2ul (1:100d), 0.4ul (1:100d), 1ul (1:100d); 50C, 10min, 70C, 20min
  • PCR with blue/orange (mixed primers tube and new Kapa master mix)

QPCR curve[edit]

None treatment File:8.17.11 regularHap.jpg

+0.2ul protease File:8.17.11 0.2ul.jpg

+0.4ul protease File:8.17.11 0.4ul.jpg

+1ul protease File:8.17.11 1ul.jpg


TBE PAGE check[edit]

File:8.18.11 0-0.2.jpg

File:8.18.11 0.4-1.jpg

Thoughts[edit]

  • Protease treatments greatly helps to control unspecific amplification!
  • Protease treatments seems to increase target amplification.
  • Questions: 1) size shifts down 2) unevenness between samples (1ul protease seems best in this part)