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