Rui:LabNotes/Haplotyping/2011-8-23: Difference between revisions
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Set1: 1-12 | Set1: 1-12 | ||
[[File:8.23.11_set1.jpg]] | [[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 | Set2: 1-12 | ||
[[File:8.23.11_set2.jpg]] | [[File:8.23.11_set2.jpg]] |
Revision as of 16:41, 2 September 2011
Haplotyping in tube
- Protocol as in lab note on 8.17 and 8.18 [1]
Samples
- cells aliquot on 8/8/11 [2]
Procedure
- 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
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
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