Noi/NOTES/2014-8-27

From ZhangLabWiki
Jump to navigation Jump to search

BSPP LMS cluster capture of colon cancer primary tumor DNA[edit]

Calendar
Probe prep

Experimental plan[edit]

  • Use MONOD V5 set A to capture DNA from colon tumor DNA
  • Test sensitivity of BSPP capture of this probe set on 10, 20, 100 and 200ng

Bisulfite conversion and ssDNA quantification[edit]

  • I did bisulfite conversion of 5 colon cancer DNA sample in duplicates using EZ DNA Methylation-Lightning™ Kit
  • I followed the instruction provided by manufacturer
Sample ID Conc. (ng/ul) Volume for 600ng (ul) H2O to 20ul
6-T-1 43.40 13.82 6.18
6-T-2 31.60 18.99 1.01
6-T-3 31.50 19.05 0.95
6-T-4 41.70 14.39 5.61
6-T-5 30.50 19.67 0.33
1.    98°C  for 8 minutes
2.    54°C  for 60 minutes
3.    Continue to desulfonation
- Elute with 18ul Elution Buffer
- The volume left after Elution ~16ul
- Quantify concentration and recovery yield by Qubit ssDNA assay kit
Sample Conc. in the Qubit Unit uL used Dilution Conc. (ng/ul) Yield in 16ul (ng) % Recovery
6-T-1_1 136 ng/mL 1 200 27.20 435.20 72.53
6-T-1_2 144 ng/mL 1 200 28.90 462.40 77.07
6-T-2_1 92.2 ng/mL 1 200 18.40 294.40 49.07
6-T-2_2 95 ng/mL 1 200 19.00 304.00 50.67
6-T-3_1 137 ng/mL 1 200 27.40 438.40 73.07
6-T-3_2 131 ng/mL 1 200 26.30 420.80 70.13
6-T-4_1 117 ng/mL 1 200 23.40 374.40 62.40
6-T-4_2 134 ng/mL 1 200 26.80 428.80 71.47
6-T-5_1 133 ng/mL 1 200 26.60 425.60 70.93
6-T-5_2 140 ng/mL 1 200 27.90 446.40 74.40

BSPP capture set up[edit]

Probe calculation[edit]

Probe:target 200 : 1 Unit, calculation
Probe size 73,574
Template 10 ng
Human gDNA MW 1.950E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (10ng) 5.128E-21 mol (10ng/1.950E+12 g/mol)
Probe required (200:1) 1.026E-18 mol (2.5641E-20 * 200)
Probe MW (73,574 probes, 110nt) 2.636E+09 g/mol (73,574 probes * (110nt * 325Da/nt + 79Da))
Amount probe required 2.704E-09 g
Amount probe required 2.7 ng
Input bis-DNA (ng) MONOD V5 set A amount (ng)
10 2.70
20 5.40
100 27.00
200 54.00