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Rui:LabNotes/Haplotyping/2011-11-4
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==Test condition for haplotyping== * Continue with lab note 11.3.11 * Tagmentation and PCR ===Samples=== * Non-MDA ~1pg cell lysates: from same patch of cells as MDA samples, Ptnase treatment, Nextera * MDA samples: 1ul each of all 8 MDA product [17ct-2, 22ct-3, 27ct-3,4, 30ct-1,2,3,4] ranging from 1ng - 20ng ===Procedure=== * Tagmentation: 1ul 1:50d Trp-I, 1ul LMW buffer, fill up to 5ul * PCR: 15ul Kapa QPCR MM, 0.3ul each B/O primer, fill up to 30ul ===Results=== MDA samples [[File:11.4.11_MDA-nextera.jpg|400px]] non-MDA samples [[File:11.4.11_ctrls.jpg|400px]] gel check: MDA samples are over-amplified except 2 from 27ct[[File:11.4.11_gel.jpg|300px]] ==Simplify protocol== * Skip purification after MDA * Combine 2 rounds of PCR ===Samples=== * 10ng jurkat + 1ul dALS + 1ul dNS + 7ul MDA MM --> take 3ul for tagmentation * 1ul each of all 8 MDA product [17ct-2, 22ct-3, 27ct-3,4, 30ct-1,2,3,4] ranging from 1ng - 20ng ===Procedure=== * 3ng jurkat in 10ul MDA MM --> 65C, 3min * 5ul tagmentation reaction * 30ul PCR: 15ul Kapa QPCR, 0.6ul Adaptor 1, 0.6ul barcoded-adaptor 2 (GA-A2 Indx73-80) * 58C, 4 cycles; 62C, 8 cycles ===Results=== * Consistent with previous curve, suggesting 1 step PCR works * Jurkat gDNA directly with MDA MM seems fine with tagmentation and PCR, suggesting purification can skip 58C for 4 cycles [[File:11.4.11_58C.jpg|400px]] 62C for 8 cycles [[File:11.4.11_62C.jpg|400px]] gel check after beads purification [[File:11.7.11_gel.jpg|300px]]
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