Rui:LabNotes/Haplotyping/2011-8-23

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Revision as of 16:59, 2 September 2011 by >RuiLiu (→‎TBE PAGE)
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Haplotyping in tube[edit]

  • Protocol as in lab note on 8.17 and 8.18 [1]

Samples[edit]

  • cells aliquot on 8/8/11 [2]

Procedure[edit]

  1. 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
  2. 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
  3. Tagmentation with 0.4ul of 1:50d transposon
  4. Protease treatment: 1ul (1:100d and then 1:5d); 50C, 10min, 70C, 20min
  5. 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.
  6. 25ul beads mix and RT. 8min, stand for 5min, take off sup. as much as possible
  7. 2nd round of PCR with 50ul Kapa HF and Adaptor 1/Adaptor 2 (barcode 1-12); 60C 2c and 62C 3c.
  8. 90ul beads mix and purification

QPCR curve[edit]

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[edit]

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[edit]

  • Processed by Matt
  • He met a trouble of filtering gels by Nanosep column, somehow there were not much gels on the column...
' [] estimate Set1 (hap1-12) [] estimate 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