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Rui:LabNotes/Haplotyping/2011-8-23
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==Haplotyping in tube== * Protocol as in lab note on 8.17 and 8.18 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Haplotyping/2011-8-17] ===Samples=== * cells aliquot on 8/8/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Haplotyping/2011-8-8] ===Procedure=== # 1 ul of 10 cells/ul + 60ul lysis buffer (1X, made on 8/3/11 [2]); immediately split in 2 tubes (set1 and set2); 37C, 30min # Add 0.3ul protease to tube, immediately aliquot 1ul/tube for 12 tubes from set 1 and 12 tubes from set2; 50C, 15min, 70C, 15min # Tagmentation with 0.4ul of 1:50d transposon # Protease treatment: 1ul (1:100d and then 1:5d); 50C, 10min, 70C, 20min # 1st round of PCR with blue/orange (mixed primers tube and Kapa master mix); 30ul total; set for 22 cycles until NTC coming up. 3 samples (set1.10, set1.12, and set2.9) stopped at 16 cycles. # 25ul beads mix and RT. 8min, stand for 5min, take off sup. as much as possible # 2nd round of PCR with 50ul Kapa HF and Adaptor 1/Adaptor 2 (barcode 1-12); 60C 2c and 62C 3c. # 90ul beads mix and purification ===QPCR curve=== set1: #1-6 [[File:8.23.11_set1.1-6.jpg]] set1: #7-12 [#10(4 in the pic.) and #12 (6 in the pic) were stopped at 16c] [[File:8.23.11_set1.7-12.jpg]] set2: #1-6 [[File:8.23.11_set2.1-6.jpg]] set2: #7-12 [#9(3 in the pic.) was stopped at 16c] [[File:8.23.11_set2.7-12.jpg]] ===TBE PAGE=== gel check for comparison of 2 rounds of PCR [[File:8.23.11_2 PCRs.jpg]] Set1: 1-12 [[File:8.23.11_set1.jpg]] Repeat 7-12 (for last run, somehow, #9 was not taken.) [[File:8.23.11_set1-9.jpg]] Set2: 1-12 [[File:8.23.11_set2.jpg]] ===Size selection and final check=== * Processed by Matt * He met a trouble of filtering gels by Nanosep column, somehow there were not much gels on the column... {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''[] estimate''' | align="center" style="background:#f0f0f0;"|'''Set1 (hap1-12)''' | align="center" style="background:#f0f0f0;"|'''[] estimate''' | align="center" style="background:#f0f0f0;"|'''Set2 (hap13-23)''' |- | 1||66.57||1.97||36.37||1.69 |- | 2||32.9||3.99||12.31||5 |- | 3||84.11||1.56||28.35||2.17 |- | 4||123.75||1.06||53.38||1.15 |- | 5||117.93||1.11||25.01||2.46 |- | 6||90.44||1.45||27.4||2.25 |- | 7||65.66||2.36||13.89||4.43 |- | 8||157.87||0.59||44.09||1.4 |- | 9||87.6||1.81|||| |- | 10||160.19||0.8||31.43||1.96 |- | 11||47.48||4||25.61||2.4 |- | 12||71.35||2.31||12.5||4.92 |- | ||||23.03||||29.84 |- | |} Final gel check
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