Rui:LabNotes/Haplotyping/2011-9-15

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Test for Nextera kit (Trp-Roche)[edit]

Cell samples[edit]

  • Alan gave me 2 cell patches: w/o or w/ colcemid treatment
  • GM12878 P9 - 556,449 cells/ml; GM12878 p9 - 865,509 cells/ml
  • Collect, wash, resuspend and dilute to 10cell/ul
  • Aliquot 1ul/tube and store at -80C

Procedure[edit]

  • Aliquot before adding 1ul Ptnase (1:100d)
  • Test Trp-R
  • Test Trp-B/O and Trp-P/P primer sets
  • Test different Ta
Cell lysis and aliquot ' ' Notes: '
1 tube with 1ul of 10 cells/ul Try to reduce physical force and lysis time
add 60ul 1X lysis buffer, mixed No pipetting, gently stir by P20 tip
37C, 5-10min
Protease treatment
add 0.6ul protease Aliquot before protease addition
mix and aliquot to 24 tubes Aliquot by using P20 tip
50C, 15min and try to use contineous gun
70C, 15min
Tagmentation
DNA 2 Using Trp-Roche
5X LMWbuffer 1 Trp-I as ctrl
1:50 dilted enzyme 0.4
H2O 1.6
total 5
55C, 5min
Protease treatment
dilute protease to 1:500
add 1ul to each tube
50C, 10min
70C, 20min
1st round of PCR Using Trp-B/O primer set (R1/R2)
DNA 6 Using Trp-P/P primer set
Kapa fast mix 15 Test different Ta
Orange (10uM) 0.24 55C, 52C, 50C, 48C
blue (10uM) 0.24
H2O 8.52
Total 30
72C, 3min
95C 30sec, [95C 10sec; 58C 30sec; 72C 2min] X 20 58C
72C, 3min

results[edit]

lab note on 9/16/11