Noi/NOTES/2013-7-19: Difference between revisions
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* 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. | * 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. | * Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion. | ||
== BSPP capture set up (plan) == | |||
==== Original set ==== | |||
* Conc.: 25.4 ng/ul (by Qubit ssDNA assay) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe:target''' | |||
| align="center" style="background:#f0f0f0;"|'''500:1''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Probe size||12000|| | |||
|- | |||
| Template||150||ng | |||
|- | |||
| Human gDNA MW||1.9500E+12||g/mol | |||
|- | |||
| Human gDNA (150ng)||7.6923E-20||mol (150ng/1.9500E+12 g/mol) | |||
|- | |||
| Probe required (500:1)||3.8462E-17||mol (7.6923E-20 mol * 500) | |||
|- | |||
| Probe MW (12,000 probes, 110nt)||4.2900E+08||g/mol (12000 probes * 110nt * 325Da/nt) | |||
|- | |||
| Amount probe required||1.6500E-08||g (4.2900E+08 g/mol * 3.8462E-17 mol) | |||
|- | |||
| Amount probe required||16.50||ng | |||
|} | |||
==== Subset A ==== | |||
* Conc.: 17.6ng/ul (by Qubit ssDNA assay) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe:target''' | |||
| align="center" style="background:#f0f0f0;"|'''500:1''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Probe size||566||probes | |||
|- | |||
| Template||150||ng | |||
|- | |||
| Human gDNA MW||1.9500E+12||g/mol (3E+09 * 650Da/bp+157.9Da) | |||
|- | |||
| Human gDNA (200ng)||7.6923E-20||mol (200ng/1.9500E+12 g/mol) | |||
|- | |||
| Probe required (500:1)||3.8462E-17||mol (1.5385E-19mol * 500) | |||
|- | |||
| Probe MW (566 probes, 1100nt)||2.0235E+07||g/mol (566 probes * 110nt * 325Da/nt) | |||
|- | |||
| Amount probe required||7.7825E-10||g 2.0235E+07 g/mol * 3.8462E-17mol) | |||
|- | |||
| Amount probe required||0.78||ng | |||
|} | |||
* So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 8.89ng | |||
==== Subset B ==== | |||
* Conc.: 57.9ng/ul (by Qubit ssDNA assay) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe:target''' | |||
| align="center" style="background:#f0f0f0;"|'''500:1''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Probe size||11397||probes | |||
|- | |||
| Template||150||ng | |||
|- | |||
| Human gDNA MW||1.9500E+12||g/mol (3E+09 * 650Da/bp+157.9Da) | |||
|- | |||
| Human gDNA (200ng)||7.6923E-20||mol (150ng/1.9500E+12 g/mol) | |||
|- | |||
| Probe required (500:1)||3.8462E-17||mol (1.5385E-19mol * 500) | |||
|- | |||
| Probe MW (11,397 probes, 1100nt)||4.0744E+08||g/mol (11397 probes * 110nt * 325Da/nt) | |||
|- | |||
| Amount probe required||1.5671E-08||g (4.0744E+08 g/mol * 3.8462E-17 mol) | |||
|- | |||
| Amount probe required||15.67||ng | |||
|} | |||
* So for 1:5:5 (Original:A:B), amount probe required = 0.15.67ng * 5 = 78.35ng |
Revision as of 17:45, 20 July 2013
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.
BSPP capture set up (plan)
Original set
- Conc.: 25.4 ng/ul (by Qubit ssDNA assay)
Probe:target | 500:1 | ' |
Probe size | 12000 | |
Template | 150 | ng |
Human gDNA MW | 1.9500E+12 | g/mol |
Human gDNA (150ng) | 7.6923E-20 | mol (150ng/1.9500E+12 g/mol) |
Probe required (500:1) | 3.8462E-17 | mol (7.6923E-20 mol * 500) |
Probe MW (12,000 probes, 110nt) | 4.2900E+08 | g/mol (12000 probes * 110nt * 325Da/nt) |
Amount probe required | 1.6500E-08 | g (4.2900E+08 g/mol * 3.8462E-17 mol) |
Amount probe required | 16.50 | ng |
Subset A
- Conc.: 17.6ng/ul (by Qubit ssDNA assay)
Probe:target | 500:1 | ' |
Probe size | 566 | probes |
Template | 150 | ng |
Human gDNA MW | 1.9500E+12 | g/mol (3E+09 * 650Da/bp+157.9Da) |
Human gDNA (200ng) | 7.6923E-20 | mol (200ng/1.9500E+12 g/mol) |
Probe required (500:1) | 3.8462E-17 | mol (1.5385E-19mol * 500) |
Probe MW (566 probes, 1100nt) | 2.0235E+07 | g/mol (566 probes * 110nt * 325Da/nt) |
Amount probe required | 7.7825E-10 | g 2.0235E+07 g/mol * 3.8462E-17mol) |
Amount probe required | 0.78 | ng |
- So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 8.89ng
Subset B
- Conc.: 57.9ng/ul (by Qubit ssDNA assay)
Probe:target | 500:1 | ' |
Probe size | 11397 | probes |
Template | 150 | ng |
Human gDNA MW | 1.9500E+12 | g/mol (3E+09 * 650Da/bp+157.9Da) |
Human gDNA (200ng) | 7.6923E-20 | mol (150ng/1.9500E+12 g/mol) |
Probe required (500:1) | 3.8462E-17 | mol (1.5385E-19mol * 500) |
Probe MW (11,397 probes, 1100nt) | 4.0744E+08 | g/mol (11397 probes * 110nt * 325Da/nt) |
Amount probe required | 1.5671E-08 | g (4.0744E+08 g/mol * 3.8462E-17 mol) |
Amount probe required | 15.67 | ng |
- So for 1:5:5 (Original:A:B), amount probe required = 0.15.67ng * 5 = 78.35ng