Rui:LabNotes/Haplotyping/2011-6-4

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Revision as of 20:32, 5 June 2011 by >Sam Chiang (Created page with '==Repeat Haplotyping== * 5 cells collected by Andy * Lysis buffer w/ NP40 (Alice's) * Newly opennd Qiagen Protease (5.11.11) * Increase transposon from 0.2ul to 0.4ul * Slightly …')
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Repeat Haplotyping

  • 5 cells collected by Andy
  • Lysis buffer w/ NP40 (Alice's)
  • Newly opennd Qiagen Protease (5.11.11)
  • Increase transposon from 0.2ul to 0.4ul
  • Slightly decrease orange/blue from 100nM to 80nM

Procedure

  1. Add 30ul lysis buffer to 5 cells, mixed, 37C, 15min
  2. Add 0.3ul Protease, mixed, aliquot into 30 tubes (1ul/tube)
  3. Incubate at 50C, 10min and then 70C, 10min
  4. Tagmentation: 1ul LMW buffer, 0.4ul 1:50 diluted transposon in total 5ul; 55C, 5min (Add NTC1 for tagmentation)
  5. Kapa PCR: 0.2ul orange/blue primer each in total 25ul (Add NTC2 for PCR); 28 cycles

QPCR curve

All are amplified from cycle 20, #5 starts from cycle 15 (peak at 23 cycles, down to NTC level at 28 cycles)

File:6.4.11 hap QPCR.jpg