Noi/NOTES/2013-7-19: Difference between revisions
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>Noi m (→Subset B) |
>Noi mNo edit summary |
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* Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette | * Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette | ||
* Mix 15 time by pipetting | * Mix 15 time by pipetting | ||
* Add 40ul of mineral oil | * Add 40ul of mineral oil & seal with silicone sealing mat | ||
* Spin the plate and place on thermocycler | * Spin the plate and place on thermocycler | ||
'''Program''' | |||
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h | |||
-> add 2ul SLN mix (10x Titanium Taq; 0.5U/ul AmpLigase; 100uM dNTP) | |||
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. | |||
==== SLN mix solution ==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Stock conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Prepare volume (ul), total 80ul''' | |||
|- | |||
| Titanium Taq (Klentaq fragment)||10||U/ul||2||U/ul||16.00 | |||
|- | |||
| AmpLigase||5||U/ul||0.5||U/ul||8.00 | |||
|- | |||
| dNTP||1||mM||100||uM||8.00 | |||
|- | |||
| 10x AmpLigase Buffer||10||x||1||x||8.00 | |||
|- | |||
| H2O||||||||||40.00 | |||
|} | |||
* Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette. | |||
* Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment. |
Revision as of 22:13, 21 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 = 15.67ng * 5 = 78.35ng
Summary of probes required for cpature
Probe set | Conc. (ng/ul) | Vmount required | Volume (ul)/samples | Volume for ~38x rxn |
Original | 25.40 | 16.50 | 0.65 | 24.69 |
Subset A | 17.60 | 3.89 | 0.22 | 8.40 |
Subset B | 57.90 | 78.35 | 1.35 | 51.42 |
- I will do two captures for positive control (12878) to see the consistency between the two technical replicates and one NTC
- I will save at least 2ul of bis-cvt DNA for bisulfite PCR if we need to do that. However, Dr. Zhang suggested us to ignore the recommended_4 site.
Sample IDs | WellIDs | Conc. (ng/ul) | Volume required (ul) | BP-O (ul) | BP.norm-A (ul) | BP.norm-B (ul) | 10X Ampligase Buffer (ul) |
H2O (ul) | Total vulome (ul) | Total biscvt DNA (ng) |
1 | A1 | 21.00 | 7.14 | 0.65 | 0.22 | 1.35 | 2.00 | 8.64 | 20.00 | 150.00 |
2 | B1 | 24.40 | 6.15 | 0.65 | 0.22 | 1.35 | 2.00 | 9.63 | 20.00 | 150.00 |
3 | C1 | 19.10 | 7.85 | 0.65 | 0.22 | 1.35 | 2.00 | 7.93 | 20.00 | 150.00 |
4 | D1 | 22.60 | 6.64 | 0.65 | 0.22 | 1.35 | 2.00 | 9.14 | 20.00 | 150.00 |
5 | E1 | 20.60 | 7.28 | 0.65 | 0.22 | 1.35 | 2.00 | 8.50 | 20.00 | 150.00 |
6 | F1 | 21.20 | 7.08 | 0.65 | 0.22 | 1.35 | 2.00 | 8.70 | 20.00 | 150.00 |
7 | G1 | 22.00 | 6.82 | 0.65 | 0.22 | 1.35 | 2.00 | 8.96 | 20.00 | 150.00 |
8 | H1 | 22.20 | 6.76 | 0.65 | 0.22 | 1.35 | 2.00 | 9.02 | 20.00 | 150.00 |
9 | A2 | 20.60 | 7.28 | 0.65 | 0.22 | 1.35 | 2.00 | 8.50 | 20.00 | 150.00 |
10 | B2 | 24.00 | 6.25 | 0.65 | 0.22 | 1.35 | 2.00 | 9.53 | 20.00 | 150.00 |
11 | C2 | 19.06 | 7.87 | 0.65 | 0.22 | 1.35 | 2.00 | 7.91 | 20.00 | 150.00 |
12 | D2 | 18.78 | 7.99 | 0.65 | 0.22 | 1.35 | 2.00 | 7.79 | 20.00 | 150.00 |
13 | E2 | 2.06 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 26.78 |
14 | F2 | 8.08 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 105.04 |
15 | G2 | 6.50 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 84.50 |
16 | H2 | 12.32 | 12.18 | 0.65 | 0.22 | 1.35 | 2.00 | 3.60 | 20.00 | 150.00 |
17 | A3 | 11.56 | 12.98 | 0.65 | 0.22 | 1.35 | 2.00 | 2.80 | 20.00 | 150.00 |
18 | B3 | 12.24 | 12.25 | 0.65 | 0.22 | 1.35 | 2.00 | 3.53 | 20.00 | 150.00 |
19 | C3 | 11.44 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 148.72 |
20 | D3 | 7.92 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 102.96 |
21 | E3 | 8.10 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 105.30 |
22 | F3 | 11.38 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 147.94 |
23 | G3 | 13.96 | 10.74 | 0.65 | 0.22 | 1.35 | 2.00 | 5.04 | 20.00 | 150.00 |
24 | H3 | 19.72 | 7.61 | 0.65 | 0.22 | 1.35 | 2.00 | 8.17 | 20.00 | 150.00 |
25 | A4 | 21.20 | 7.08 | 0.65 | 0.22 | 1.35 | 2.00 | 8.70 | 20.00 | 150.00 |
26 | B4 | 23.00 | 6.52 | 0.65 | 0.22 | 1.35 | 2.00 | 9.26 | 20.00 | 150.00 |
27 | C4 | 23.80 | 6.30 | 0.65 | 0.22 | 1.35 | 2.00 | 9.48 | 20.00 | 150.00 |
28 | D4 | 31.40 | 4.78 | 0.65 | 0.22 | 1.35 | 2.00 | 11.00 | 20.00 | 150.00 |
29 | E4 | 20.80 | 7.21 | 0.65 | 0.22 | 1.35 | 2.00 | 8.57 | 20.00 | 150.00 |
30 | F4 | 10.46 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 135.98 |
31 | G4 | 47.00 | 3.19 | 0.65 | 0.22 | 1.35 | 2.00 | 12.59 | 20.00 | 150.00 |
32 | H4 | 56.00 | 2.68 | 0.65 | 0.22 | 1.35 | 2.00 | 13.10 | 20.00 | 150.00 |
33 | A5 (12878) | 24.00 | 6.25 | 0.65 | 0.22 | 1.35 | 2.00 | 9.53 | 20.00 | 150.00 |
34 | B5 (12878) | 24.60 | 6.10 | 0.65 | 0.22 | 1.35 | 2.00 | 9.68 | 20.00 | 150.00 |
35 | C5 (NTC) | 0.00 | 0.00 | 0.65 | 0.22 | 1.35 | 2.00 | 15.78 | 20.00 | 0.00 |
Reaction mix
Components | 1x rxn | 38 rxn mix |
10X Ampligase buffer | 2.00 | 76.00 |
BP-O | 0.65 | 24.70 |
BP.norm-A | 0.22 | 8.36 |
BP.norm-B | 1.35 | 51.30 |
Total | 4.22 | 160.36 |
- Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette
- Mix 15 time by pipetting
- Add 40ul of mineral oil & seal with silicone sealing mat
- Spin the plate and place on thermocycler
Program -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix (10x Titanium Taq; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN mix solution
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (ul), total 80ul |
Titanium Taq (Klentaq fragment) | 10 | U/ul | 2 | U/ul | 16.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 8.00 |
dNTP | 1 | mM | 100 | uM | 8.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 8.00 |
H2O | 40.00 |
- Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette.
- Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment.