Noi/NOTES/2013-7-19

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DNA methylation assay for Blueprint project

Bisulfite conversion

  • I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
Sample IDs WellIDs Conc. (ng/ul) Volume (ul) H2O (ul) CT conversion reagent (ul) Total volume (ul) Total amount (ng)
1 A1 37.71 13.26 6.74 130.00 150.00 500.00
2 B1 39.43 12.68 7.32 130.00 150.00 500.00
3 C1 50.57 9.89 10.11 130.00 150.00 500.00
4 D1 34.86 14.34 5.66 130.00 150.00 500.00
5 E1 56.00 8.93 11.07 130.00 150.00 500.00
6 F1 44.29 11.29 8.71 130.00 150.00 500.00
7 G1 73.71 6.78 13.22 130.00 150.00 500.00
8 H1 29.71 16.83 3.17 130.00 150.00 500.00
9 A2 40.29 12.41 7.59 130.00 150.00 500.00
10 B2 33.43 14.96 5.04 130.00 150.00 500.00
11 C2 44.86 11.15 8.85 130.00 150.00 500.00
12 D2 52.86 9.46 10.54 130.00 150.00 500.00
**13 E2 11.34 9.00 11.00 130.00 150.00 102.09
*14 F2 36.86 9.00 11.00 130.00 150.00 331.71
**15 G2 26.74 9.00 11.00 130.00 150.00 240.69
*16 H2 50.00 9.00 11.00 130.00 150.00 450.00
17 A3 34.00 14.71 5.29 130.00 150.00 500.00
18 B3 31.14 16.06 3.94 130.00 150.00 500.00
19 C3 26.46 18.90 1.10 130.00 150.00 500.00
*20 D3 20.29 20.00 0.00 130.00 150.00 405.71
*21 E3 18.23 20.00 0.00 130.00 150.00 364.57
22 F3 29.43 16.99 3.01 130.00 150.00 500.00
23 G3 47.71 10.48 9.52 130.00 150.00 500.00
24 H3 32.57 15.35 4.65 130.00 150.00 500.00
25 A4 29.14 17.16 2.84 130.00 150.00 500.00
*26 B4 19.60 20.00 0.00 130.00 150.00 392.00
27 C4 24.31 20.00 0.00 130.00 150.00 486.29
28 D4 23.66 20.00 0.00 130.00 150.00 473.14
29 E4 47.43 10.54 9.46 130.00 150.00 500.00
30 F4 76.86 6.51 13.49 130.00 150.00 500.00
31 G4 40.00 12.50 7.50 130.00 150.00 500.00
32 H4 32.29 15.49 4.51 130.00 150.00 500.00
33 A5 (12878) 58.00 8.62 11.38 130.00 150.00 500.00
34 B5 (12878) 59.00 8.47 11.53 131.00 151.00 500.00
 Program 
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
  • Eluted bisulfite converted DNA form the bead with 20ul H2O
  • I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.

ssDNA Qubit assay result

Sample IDs WellIDs Start amount (ng) Conc. (ng/ul) Yield in 17ul (ng) Yield left after
Qubit (15ul)
% Recovery in 17ul
1 A1 500.00 21.00 357.00 315.00 71.40
2 B1 500.00 24.40 414.80 366.00 82.96
3 C1 500.00 19.10 324.70 286.50 64.94
4 D1 500.00 22.60 384.20 339.00 76.84
5 E1 500.00 20.60 350.20 309.00 70.04
6 F1 500.00 21.20 360.40 318.00 72.08
7 G1 500.00 22.00 374.00 330.00 74.80
8 H1 500.00 22.20 377.40 333.00 75.48
9 A2 500.00 20.60 350.20 309.00 70.04
10 B2 500.00 24.00 408.00 360.00 81.60
11 C2 500.00 19.06 324.02 285.90 64.80
12 D2 500.00 18.78 319.26 281.70 63.85
13 E2 102.09 2.06 35.02 30.90 34.30
14 F2 331.71 8.08 137.36 121.20 41.41
15 G2 240.69 6.50 110.50 97.50 45.91
16 H2 450.00 12.32 209.44 184.80 46.54
17 A3 500.00 11.56 196.52 173.40 39.30
18 B3 500.00 12.24 208.08 183.60 41.62
19 C3 500.00 11.44 194.48 171.60 38.90
20 D3 405.71 7.92 134.64 118.80 33.19
21 E3 364.57 8.10 137.70 121.50 37.77
22 F3 500.00 11.38 193.46 170.70 38.69
23 G3 500.00 13.96 237.32 209.40 47.46
24 H3 500.00 19.72 335.24 295.80 67.05
25 A4 500.00 21.20 360.40 318.00 72.08
26 B4 392.00 23.00 391.00 345.00 99.74
27 C4 486.29 23.80 404.60 357.00 83.20
28 D4 473.14 31.40 533.80 471.00 112.82
29 E4 500.00 20.80 353.60 312.00 70.72
30 F4 500.00 10.46 177.82 156.90 35.56
31 G4 500.00 47.00 799.00 705.00 159.80
32 H4 500.00 56.00 952.00 840.00 190.40
33 A5 (12878) 500.00 24.00 408.00 360.00 81.60
34 B5 (12878) 500.00 24.60 418.20 369.00 83.64
  • There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
  • Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.