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Rui:LabNotes/Haplotyping/2011-6-4
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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=== # 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=== 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]] ===TBE PAGE=== After bead purification: 0.7X (25ul + 18ul), elute in 10ul, 1ul for gel check [[File:6.6.11_hap 1.jpg]] [[File:6.6.11_hap 2.jpg]]
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