Noi/NOTES/2014-6-16
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Preparation of RRBS sequencing libraries of solid tumor tissue sample from pancreatic cancer patients
Sample info
Project ID | Zhang lab ID | Conc. (ng/ul) |
UCSD-004-04 | PC-T-1 | 82.70 |
UCSD-004-05 | PC-T-2 | 35.80 |
UCSD-004-07 | PC-T-4 | 42.80 |
UCSD-004-09 | PC-T-6 | 15.20 |
UCSD-004-10 | PC-T-7 | 16.30 |
MspI digestion
- Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 1h
MspI digestion set up
Strip1 | PC-T-1_r1 | PC-T-1_r2 | PC-T-2_r1 | PC-T-2_r2 | PC-T-4_r1 | PC-T-4_r2 | PC-T-6_r1 | PC-T-6_r2 |
DNA conc. (ng/ul) | 82.70 | 82.70 | 35.80 | 35.80 | 42.80 | 42.80 | 15.20 | 15.20 |
Volume for 100ng (ul) | 1.21 | 1.21 | 2.79 | 2.79 | 2.34 | 2.34 | 6.58 | 6.58 |
H2O (ul) | 13.79 | 13.79 | 12.21 | 12.21 | 12.66 | 12.66 | 8.42 | 8.42 |
10X Tango Buffer | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 |
MspI | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 |
H2O in MspI Mix | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
Total (ul) | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 |
Strip2 | PC-T-7_r1 | PC-T-7_r2 | NTC |
DNA conc. (ng/ul) | 16.30 | 16.30 | 0.00 |
Volume for 100ng (ul) | 6.13 | 6.13 | 0.00 |
H2O (ul) | 8.87 | 8.87 | 15.00 |
10X Tango Buffer | 3.00 | 3.00 | 3.00 |
MspI | 2.00 | 2.00 | 2.00 |
H2O in MspI Mix | 10.00 | 10.00 | 10.00 |
Total (ul) | 30.00 | 30.00 | 30.00 |
Prep
- - UV strip tube for reaction set up
- - Aliquot H2O in 1.5mL tube & UV
- - UV 1.5mL tube for MspI reaction Mix
Msp I reaction Mix
Components | 1x rxn | 9.5 rxn Mix |
MspI (10U/ul) | 2.00 | 19.00 |
10x Tango Buffer | 3.00 | 28.50 |
H2O | 10.00 | 95.00 |
Total | 15.00 |
Procedures
- - Add H2O to each tube
- - Add 100ng of DNA to each tube
- - Add 15ul of MspI reaction Mix to each tube and pipette 5x
- - Mix by gently vortexing on PCR rack 10x
- - Spin down the tube
- - In program Noi: MspI: 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)