Rui:LabNotes/Haplotyping/2011-11-4

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Test condition for haplotyping[edit]

  • Continue with lab note 11.3.11
  • Tagmentation and PCR

Samples[edit]

  • 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[edit]

  • 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[edit]

MDA samples File:11.4.11 MDA-nextera.jpg non-MDA samples File:11.4.11 ctrls.jpg

gel check: MDA samples are over-amplified except 2 from 27ctFile:11.4.11 gel.jpg

Simplify protocol[edit]

  • Skip purification after MDA
  • Combine 2 rounds of PCR

Samples[edit]

  • 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[edit]

  • 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[edit]

  • 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 62C for 8 cycles File:11.4.11 62C.jpg

gel check after beads purification File:11.7.11 gel.jpg