Rui:LabNotes/Haplotyping/2011-6-4
Jump to navigation
Jump to search
Repeat Haplotyping[edit]
- 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[edit]
- Add 30ul lysis buffer to 5 cells, mixed, 37C, 15min
- Add 0.3ul Protease, mixed, aliquot into 30 tubes (1ul/tube)
- Incubate at 50C, 10min and then 70C, 10min
- Tagmentation: 1ul LMW buffer, 0.4ul 1:50 diluted transposon in total 5ul; 55C, 5min (Add NTC1 for tagmentation)
- Kapa PCR: 0.2ul orange/blue primer each in total 25ul (Add NTC2 for PCR); 28 cycles
QPCR curve[edit]
All are amplified from cycle 20, #5 starts from cycle 15 (peak at 23 cycles, down to NTC level at 28 cycles)
TBE PAGE[edit]
After bead purification: 0.7X (25ul + 18ul), elute in 10ul, 1ul for gel check