Rui:LabNotes/Haplotyping/2011-8-23
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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 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[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