Noi/NOTES/2014-5-29

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MONOD V1N3 BSPP capture

Link to calendar: [[1]]

  • I will do BSPP capture using the new probe set, MONOD V1N3. This probe set, Dr. Zhang selected the probes with high capture efficiency from GP1V4 and MONOD V3. The size of probe is quite small ~2400 probes.
  • I am going to test capture sensitivity on different amount of BIS-JURKAT DNA input at 5, 10, 25, and 50ng with 200ng as positive control
  • To test the recovery rate after bisulfite conversion, I perform bisulfite conversion at different amount of gDNA.

Bisulfite conversion

  • Received new tube of JURKAT DNA.
  • Re-quantified concentration with Qubit dsDNA assay HS (1ul in 200ul reaction = 200X dilution)
    • Conc. = 82.1ng/ul
  • Amount/ # of tube for bisulfite conversion
    • 100ng/ 3tubes
    • 200ng/ 3tubes
    • 600ng/ 2tubes
Sample Conc. (ng/ul) Amount (ng) Volume (ul) H2O (ul) Lightnig Conversion reagent Total
JK-1 82.10 100.00 1.22 18.78 130.00 150.00
JK-2 82.10 100.00 1.22 18.78 130.00 150.00
JK-3 82.10 100.00 1.22 18.78 130.00 150.00
JK-4 82.10 200.00 2.44 17.56 130.00 150.00
JK-5 82.10 200.00 2.44 17.56 130.00 150.00
JK-6 82.10 200.00 2.44 17.56 130.00 150.00
JK-7 82.10 600.00 7.31 12.69 130.00 150.00
JK-8 82.10 600.00 7.31 12.69 130.00 150.00
  • Follow the instruction of the kit
  • Elute with 17ul Elution buffer
  • Volume left is ~15ul
  • Quantified concentration of BIS- JURKAT DNA with Qubit ssDNa assay (1ul in 200ul reaction = 200x dilution)

ssDNA Qubit assay result

Sample Conc. in the Qubit Unit uL used Dilution Sample conc. (ng/ul) Yield in 15ul (ng) Input DNA (ng) % Recovery
JK-1 15.2 ng/mL 1 200 3.03 45.45 100 45.45
JK-2 19.1 ng/mL 1 200 3.82 57.30 100 57.30
JK-3 13.3 ng/mL 1 200 2.66 39.90 100 39.90
JK-4 41 ng/mL 1 200 8.21 123.15 200 61.58
JK-5 36.6 ng/mL 1 200 7.31 109.65 200 54.83
JK-6 39.4 ng/mL 1 200 7.88 118.20 200 59.10
JK-7 133 ng/mL 1 200 26.6 399.00 600 66.50
JK-8 131 ng/mL 1 200 26.2 393.00 600 65.50
DNA input Ave. % recovery rate STDEV
100ng 47.55 8.89
200ng 58.5 3.41
600ng 66 0.71
  • It seems like recovery rate is proportional to DNA input. However, I could expect at least 40% from 100ng input. I should try with lower amount of DNA (25 or 50ng) since cell-free DNA from cancer samples have very low DNA amount and varied between different samples.

BSPP capture set up

  • BIS-DNA: 5ng, 10ng, 25ng, 50ng and 200ng as PC
  • Probe-to-target: 1,000:1 & 2,000:1

Probe capculation

Template (ng) ratio V1N3 amount required (ng)
200 1,000:1 8.80
200 2,000:1 17.61
5 1,000:1 0.22
5 2,000:1 0.44
10 1,000:1 0.44
10 2,000:1 0.88
25 1,000:1 1.10
25 2,000:1 2.20
50 1,000:1 2.20
50 2,000:1 4.40

V1N3 capture set up

  • X-XXXX = BIS-DNA input amount-probe-to-target ratio
strip 1 5-1000 5-1000 10-1000 10-1000 25-1000 25-1000 50-1000 50-1000
BIS-DNA required (ng) 5 5 10 10 25 25 50 50
JK # JK-1 JK-1 JK-2 JK-2 JK-4 JK-4 JK-5 JK-5
Conc. 3.03 3.03 3.82 3.82 8.21 8.21 7.31 7.31
Volume (ul) 1.65 1.65 2.62 2.62 3.05 3.05 6.84 6.84
H2O (ul) 13.05 13.05 11.78 11.78 10.45 10.45 5.15 5.15
V1N3 amount (ng) 0.22 0.22 0.44 0.44 1.10 1.10 2.20 2.20
V1N3 conc. (ng/ul) 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73
V1N3 volume (ul) 0.30 0.30 0.60 0.60 1.50 1.50 3.01 3.01
10X Ampligase Buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00