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)