Revision as of 02:15, 21 July 2013 by >Noi
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
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
- Spin the plate and place on thermocycler