Noi/NOTES/2011-7-28
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BSPP DMR330k capture of GA and control gDNA from blood sample[edit]
Bisulfite conversion[edit]
- Follow protocol provided with the kit.
- EZ-96 DNA Methylation-Gold Kit
- Catalog# D5007 (Shallow-Well Format)
- Note:
- Speed and time of centrifugation could be adjusted based on centrifuge model
- Used 1 or1.2 ug of gDNA in bisulfite conversion reaction to get enough amount of bis-cvt DNA for BSPP capture. This experiment used 1.2ug
- The minmum DNA concentration of gDNA is 50ng/ul.
- Elute bis-cvt gDNA from the column in the last step with 20ul of Elution buffer to get the final volume ~ 17ul (will use 1 ul for Qubit ssDNA assay)
Qubit ss-DNA assay[edit]
- Follow protocol provided with the kit
- Add 1ul of bis-cvt gDNA sample to 199ul of 1x Qubit ssDNA reagent (total assay volume = 200ul)
Sample | Conc. In Qubit (ng/mL) | Volume (ul) | Final conc (ng/ul) | Yields (ng) | Volume for 300ng | Well ID |
1.00 | 92.80 | 16.00 | 18.56 | 296.96 | 16.16 | A1 |
2.00 | 704.00 | 16.00 | 140.80 | 2252.80 | 2.13 | B1 |
3.00 | 84.00 | 16.00 | 49.40 | 790.40 | 6.07 | C1* |
4.00 | 109.00 | 16.00 | 21.80 | 348.80 | 13.76 | D1 |
5.00 | 27.00 | 16.00 | 48.40 | 774.40 | 6.20 | E1* |
6.00 | 653.00 | 16.00 | 130.60 | 2089.60 | 2.30 | F1 |
7.00 | 26.00 | 16.00 | 44.60 | 713.60 | 6.73 | G1* |
8.00 | 25.00 | 16.00 | 48.80 | 780.80 | 6.15 | H1* |
9.00 | 33.70 | 16.00 | 49.60 | 793.60 | 6.05 | A2* |
10.00 | 45.40 | 16.00 | 48.40 | 774.40 | 6.20 | B2* |
11.00 | 207.00 | 16.00 | 41.40 | 662.40 | 7.25 | C2 |
12.00 | 200.00 | 16.00 | 40.00 | 640.00 | 7.50 | D2 |
13.00 | 197.00 | 16.00 | 39.40 | 630.40 | 7.61 | E2 |
14.00 | 115.00 | 16.00 | 23.00 | 368.00 | 13.04 | F2 |
15.00 | 602.00 | 16.00 | 120.40 | 1926.40 | 2.49 | G2 |
16.00 | 234.00 | 16.00 | 46.80 | 748.80 | 6.41 | H2 |
17.00 | 39.70 | 16.00 | 46.40 | 742.40 | 6.47 | A3* |
18.00 | 104.00 | 16.00 | 20.80 | 332.80 | 14.42 | B3 |
19.00 | 278.00 | 16.00 | 55.60 | 889.60 | 5.40 | C3 |
20.00 | 109.00 | 16.00 | 21.80 | 348.80 | 13.76 | D3 |
21.00 | 59.00 | 16.00 | 11.80 | 188.80 | 25.42 | E3** |
22.00 | 132.00 | 16.00 | 26.40 | 422.40 | 11.36 | F3 |
23.00 | 12.50 | 16.00 | 44.60 | 713.60 | 6.73 | G3* |
24.00 | 207.00 | 16.00 | 41.40 | 662.40 | 7.25 | H3 |
25.00 | 156.00 | 16.00 | 31.20 | 499.20 | 9.62 | A4 |
26.00 | 174.00 | 16.00 | 34.80 | 556.80 | 8.62 | B4 |
27.00 | 209.00 | 16.00 | 41.80 | 668.80 | 7.18 | C4 |
28.00 | 75.60 | 16.00 | 42.40 | 678.40 | 7.08 | D4* |
29.00 | 156.00 | 16.00 | 31.20 | 499.20 | 9.62 | E4 |
30.00 | 163.00 | 16.00 | 32.60 | 521.60 | 9.20 | F4 |
31.00 | 220.00 | 16.00 | 44.00 | 704.00 | 6.82 | G4 |
32.00 | 193.00 | 16.00 | 38.60 | 617.60 | 7.77 | H4 |
33.00 | 440.00 | 16.00 | 88.00 | 1408.00 | 3.41 | A5 |
34.00 | 252.00 | 16.00 | 50.40 | 806.40 | 5.95 | B5 |
35.00 | 215.00 | 16.00 | 43.00 | 688.00 | 6.98 | C5 |
36.00 | 188.00 | 16.00 | 37.60 | 601.60 | 7.98 | D5 |
37.00 | 174.00 | 16.00 | 34.80 | 556.80 | 8.62 | E5 |
38.00 | 152.00 | 16.00 | 30.40 | 486.40 | 9.87 | F5 |
39.00 | 747.00 | 16.00 | 149.40 | 2390.40 | 2.01 | G5 |
40.00 | 114.00 | 16.00 | 22.80 | 364.80 | 13.16 | H5 |
41.00 | 540.00 | 16.00 | 108.00 | 1728.00 | 2.78 | A6 |
42.00 | 258.00 | 16.00 | 51.60 | 825.60 | 5.81 | B6 |
43.00 | 71.20 | 16.00 | 39.60 | 633.60 | 7.58 | C6* |
44.00 | 321.00 | 16.00 | 64.20 | 1027.20 | 4.67 | D6 |
45.00 | 187.00 | 16.00 | 37.40 | 598.40 | 8.02 | E6 |
46.00 | 167.00 | 16.00 | 33.40 | 534.40 | 8.98 | F6 |
47.00 | 326.00 | 16.00 | 65.20 | 1043.20 | 4.60 | G6 |
48.00 | 238.00 | 16.00 | 47.60 | 761.60 | 6.30 | H6 |
49.00 | 408.00 | 16.00 | 81.60 | 1305.60 | 3.68 | A7 |
50.00 | 141.00 | 16.00 | 28.20 | 451.20 | 10.64 | B7 |
51.00 | 83.10 | 16.00 | 53.40 | 854.40 | 5.62 | C7 |
52.00 | 265.00 | 16.00 | 53.00 | 848.00 | 5.66 | D7 |
53.00 | 19.70 | 16.00 | 45.00 | 720.00 | 6.67 | E7* |
54.00 | 157.00 | 16.00 | 31.40 | 502.40 | 9.55 | F7 |
55.00 | 63.10 | 16.00 | 44.80 | 716.80 | 6.70 | G7* |
56.00 | 177.00 | 16.00 | 35.40 | 566.40 | 8.47 | H7 |
57.00 | 68.20 | 16.00 | 45.80 | 732.80 | 6.55 | A8* |
58.00 | 357.00 | 16.00 | 71.40 | 1142.40 | 4.20 | B8 |
59.00 | 67.40 | 16.00 | 41.00 | 656.00 | 7.32 | C8* |
60.00 | 174.00 | 16.00 | 34.80 | 556.80 | 8.62 | D8 |
61.00 | 197.00 | 16.00 | 39.40 | 630.40 | 7.61 | E8 |
62.00 | 205.00 | 16.00 | 41.00 | 656.00 | 7.32 | F8 |
63.00 | 226.00 | 16.00 | 45.20 | 723.20 | 6.64 | G8 |
64.00 | 379.00 | 16.00 | 75.80 | 1212.80 | 3.96 | H8 |
65.00 | 278.00 | 16.00 | 55.60 | 889.60 | 5.40 | A9 |
66.00 | 359.00 | 16.00 | 71.80 | 1148.80 | 4.18 | B9 |
67.00 | 156.00 | 16.00 | 31.20 | 499.20 | 9.62 | C9 |
68.00 | 292.00 | 16.00 | 58.40 | 934.40 | 5.14 | D9 |
69.00 | 216.00 | 16.00 | 43.20 | 691.20 | 6.94 | E9 |
70.00 | 52.50 | 16.00 | 30.40 | 486.40 | 9.87 | F9* |
71.00 | 235.00 | 16.00 | 47.00 | 752.00 | 6.38 | G9 |
72.00 | 237.00 | 16.00 | 47.40 | 758.40 | 6.33 | H9 |
73.00 | 316.00 | 16.00 | 63.20 | 1011.20 | 4.75 | A10 |
74.00 | 345.00 | 16.00 | 69.00 | 1104.00 | 4.35 | B10 |
75.00 | 212.00 | 16.00 | 42.40 | 678.40 | 7.08 | C10 |
76.00 | 263.00 | 16.00 | 52.60 | 841.60 | 5.70 | D10 |
77.00 | 342.00 | 16.00 | 68.40 | 1094.40 | 4.39 | E10 |
78.00 | 219.00 | 16.00 | 43.80 | 700.80 | 6.85 | F10 |
79.00 | 220.00 | 16.00 | 44.00 | 704.00 | 6.82 | G10 |
80.00 | 304.00 | 16.00 | 60.80 | 972.80 | 4.93 | H10 |
81.00 | 119.00 | 16.00 | 23.80 | 380.80 | 12.61 | A11 |
82.00 | 303.00 | 16.00 | 60.60 | 969.60 | 4.95 | B11 |
83.00 | 247.00 | 16.00 | 49.40 | 790.40 | 6.07 | C11 |
84.00 | 161.00 | 16.00 | 32.20 | 515.20 | 9.32 | D11 |
85.00 | 143.00 | 16.00 | 28.60 | 457.60 | 10.49 | E11 |
86.00 | 337.00 | 16.00 | 67.40 | 1078.40 | 4.45 | F11 |
87.00 | 176.00 | 16.00 | 35.20 | 563.20 | 8.52 | G11 |
88.00 | 315.00 | 16.00 | 63.00 | 1008.00 | 4.76 | H11 |
89.00 | 232.00 | 16.00 | 46.40 | 742.40 | 6.47 | A12 |
90.00 | 19.30 | 16.00 | 43.00 | 688.00 | 6.98 | B12* |
91.00 | 273.00 | 16.00 | 54.60 | 873.60 | 5.49 | C12 |
92.00 | 257.00 | 16.00 | 51.40 | 822.40 | 5.84 | D12 |
93.00 | 192.00 | 16.00 | 38.40 | 614.40 | 7.81 | E12 |
94.00 | 195.00 | 16.00 | 39.00 | 624.00 | 7.69 | F12 |
95.00 | 206.00 | 16.00 | 41.20 | 659.20 | 7.28 | G12 |
96.00 | 316.00 | 16.00 | 63.20 | 1011.20 | 4.75 | H12 |
- repeat bisulfite conversion of samples with the yields < 300ng
Sample | Conc. In Qubit (ng/mL) | Volume (ul) | Final conc (ng/ul) | Yields (ng) | Volume for 300ng | Well ID |
1.00 | 247.00 | 16.00 | 49.40 | 790.40 | 6.07 | C1 |
2.00 | 242.00 | 16.00 | 48.40 | 774.40 | 6.20 | E1 |
3.00 | 223.00 | 16.00 | 44.60 | 713.60 | 6.73 | G1 |
4.00 | 244.00 | 16.00 | 48.80 | 780.80 | 6.15 | H1 |
5.00 | 248.00 | 16.00 | 49.60 | 793.60 | 6.05 | A2 |
6.00 | 242.00 | 16.00 | 48.40 | 774.40 | 6.20 | B2 |
7.00 | 232.00 | 16.00 | 46.40 | 742.40 | 6.47 | A3 |
8.00 | 55.50 | 16.00 | 11.10 | 177.60 | 27.03 | E3 |
9.00 | 223.00 | 16.00 | 44.60 | 713.60 | 6.73 | G3 |
10.00 | 212.00 | 16.00 | 42.40 | 678.40 | 7.08 | D4 |
11.00 | 198.00 | 16.00 | 39.60 | 633.60 | 7.58 | C6 |
12.00 | 267.00 | 16.00 | 53.40 | 854.40 | 5.62 | C7 |
13.00 | 225.00 | 16.00 | 45.00 | 720.00 | 6.67 | E7 |
14.00 | 224.00 | 16.00 | 44.80 | 716.80 | 6.70 | G7 |
15.00 | 229.00 | 16.00 | 45.80 | 732.80 | 6.55 | A8 |
16.00 | 205.00 | 16.00 | 41.00 | 656.00 | 7.32 | C8 |
17.00 | 152.00 | 16.00 | 30.40 | 486.40 | 9.87 | F9 |
18.00 | 215.00 | 16.00 | 43.00 | 688.00 | 6.98 | B12 |
Capture set up[edit]
Probe calculation[edit]
Subset ID | Adjusted Multiplier based on qPCR (Multiplier E) | Actual amount of probe required (probe:target ratio, 100:1) | Multiplier E x amount probe required | Probe concentration (ng/ul) | Volume (ul) | Volume for 105rxn (ul) |
DMR.s1 | 0.05 | 2.24 | 0.11 | 1.86 | 0.06 | 6.32 |
DMR.s2 | 0.07 | 2.24 | 0.16 | 1.95 | 0.08 | 8.77 |
DMR.s3 | 0.23 | 18.90 | 4.41 | 23.20 | 0.19 | 19.94 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 23.20 | 0.81 | 85.54 |
DMR.s5** | 4.83 | 18.90 | 91.29 | 29.20 | 3.13 | 328.26 |
DMR.Exp1 | 0.23 | 18.90 | 4.44 | 31.88 | 0.14 | 14.61 |
DMR.Exp2 | 0.19 | 18.90 | 3.50 | 24.11 | 0.15 | 15.26 |
DMR.Exp3 | 0.27 | 18.90 | 5.03 | 28.90 | 0.17 | 18.26 |
1.05uM Oligo suppressor-set1 | 0.15 | 15.75 | ||||
1.05uM Oligo suppressor-set2 | 0.15 | 15.75 | ||||
Total volume | 5.03 | 528.46 |
Reaction mix[edit]
Components | 1rxn (ul) | 105rxn mix (ul) |
10X AmpLigase Buffer | 2.50 | 262.50 |
DMR330k probe mix (ul) | 4.73 | 496.65 |
105nM Oligo suppressor | 0.30 | 31.50 |
Total volume (ul) | 7.53 | 790.65 |
sample ID | Final conc. (ng/ul) | Volume for 300ng(ul) | H2O (ul) | Total amount of bis-cvt gDNA (ng) | Probe mix (ul) | 10x Amligase buffer (ul) | 1.05uM Oligo suppressor set1&2 (ul) | Total volume (ul) |
A1 | 18.56 | 16.16 | 1.31 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B1 | 140.80 | 2.13 | 15.34 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C1* | 49.40 | 6.07 | 11.40 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D1 | 21.80 | 13.76 | 3.71 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E1* | 48.40 | 6.20 | 11.27 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F1 | 130.60 | 2.30 | 15.17 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G1* | 44.60 | 6.73 | 10.74 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H1* | 48.80 | 6.15 | 11.32 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A2* | 49.60 | 6.05 | 11.42 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B2* | 48.40 | 6.20 | 11.27 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C2 | 41.40 | 7.25 | 10.22 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D2 | 40.00 | 7.50 | 9.97 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E2 | 39.40 | 7.61 | 9.86 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F2 | 23.00 | 13.04 | 4.43 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G2 | 120.40 | 2.49 | 14.98 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H2 | 46.80 | 6.41 | 11.06 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A3* | 46.40 | 6.47 | 11.00 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B3 | 20.80 | 14.42 | 3.05 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C3 | 55.60 | 5.40 | 12.07 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D3 | 21.80 | 13.76 | 3.71 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E3** | 11.80 | 17.47 | 0 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F3 | 26.40 | 11.36 | 6.11 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G3* | 44.60 | 6.73 | 10.74 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H3 | 41.40 | 7.25 | 10.22 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A4 | 31.20 | 9.62 | 7.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B4 | 34.80 | 8.62 | 8.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C4 | 41.80 | 7.18 | 10.29 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D4* | 42.40 | 7.08 | 10.39 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E4 | 31.20 | 9.62 | 7.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F4 | 32.60 | 9.20 | 8.27 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G4 | 44.00 | 6.82 | 10.65 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H4 | 38.60 | 7.77 | 9.70 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A5 | 88.00 | 3.41 | 14.06 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B5 | 50.40 | 5.95 | 11.52 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C5 | 43.00 | 6.98 | 10.49 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D5 | 37.60 | 7.98 | 9.49 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E5 | 34.80 | 8.62 | 8.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F5 | 30.40 | 9.87 | 7.60 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G5 | 149.40 | 2.01 | 15.46 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H5 | 22.80 | 13.16 | 4.31 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A6 | 108.00 | 2.78 | 14.69 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B6 | 51.60 | 5.81 | 11.66 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C6* | 39.60 | 7.58 | 9.89 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D6 | 64.20 | 4.67 | 12.80 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E6 | 37.40 | 8.02 | 9.45 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F6 | 33.40 | 8.98 | 8.49 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G6 | 65.20 | 4.60 | 12.87 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H6 | 47.60 | 6.30 | 11.17 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A7 | 81.60 | 3.68 | 13.79 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B7 | 28.20 | 10.64 | 6.83 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C7 | 53.40 | 5.62 | 11.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D7 | 53.00 | 5.66 | 11.81 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E7* | 45.00 | 6.67 | 10.80 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F7 | 31.40 | 9.55 | 7.92 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G7* | 44.80 | 6.70 | 10.77 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H7 | 35.40 | 8.47 | 9.00 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A8* | 45.80 | 6.55 | 10.92 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B8 | 71.40 | 4.20 | 13.27 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C8* | 41.00 | 7.32 | 10.15 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D8 | 34.80 | 8.62 | 8.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E8 | 39.40 | 7.61 | 9.86 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F8 | 41.00 | 7.32 | 10.15 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G8 | 45.20 | 6.64 | 10.83 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H8 | 75.80 | 3.96 | 13.51 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A9 | 55.60 | 5.40 | 12.07 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B9 | 71.80 | 4.18 | 13.29 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C9 | 31.20 | 9.62 | 7.85 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D9 | 58.40 | 5.14 | 12.33 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E9 | 43.20 | 6.94 | 10.53 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F9* | 30.40 | 9.87 | 7.60 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G9 | 47.00 | 6.38 | 11.09 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H9 | 47.40 | 6.33 | 11.14 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A10 | 63.20 | 4.75 | 12.72 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B10 | 69.00 | 4.35 | 13.12 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C10 | 42.40 | 7.08 | 10.39 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D10 | 52.60 | 5.70 | 11.77 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E10 | 68.40 | 4.39 | 13.08 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F10 | 43.80 | 6.85 | 10.62 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G10 | 44.00 | 6.82 | 10.65 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H10 | 60.80 | 4.93 | 12.54 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A11 | 23.80 | 12.61 | 4.86 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B11 | 60.60 | 4.95 | 12.52 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C11 | 49.40 | 6.07 | 11.40 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D11 | 32.20 | 9.32 | 8.15 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E11 | 28.60 | 10.49 | 6.98 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F11 | 67.40 | 4.45 | 13.02 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G11 | 35.20 | 8.52 | 8.95 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H11 | 63.00 | 4.76 | 12.71 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
A12 | 46.40 | 6.47 | 11.00 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
B12* | 43.00 | 6.98 | 10.49 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
C12 | 54.60 | 5.49 | 11.98 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
D12 | 51.40 | 5.84 | 11.63 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
E12 | 38.40 | 7.81 | 9.66 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
F12 | 39.00 | 7.69 | 9.78 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
G12 | 41.20 | 7.28 | 10.19 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
H12 | 63.20 | 4.75 | 12.72 | 300.00 | 4.73 | 2.50 | 0.30 | 25.00 |
- Note:
- * = repeat bis-cvt samples
- Sample E3 have total bis-cvt amount < 300ng
- Add H2O to the 96-well plate first then add bis-cvt gDNA. Note: Try to deliver reagents to the bottom of the well
- Prepare reaction mix in 1.5 mL tube by pipetting probe, oligo suppressor and 10x AmLigase buffer and mix well.
- Aliquot reaction mix in striptube (8 tubes): 98.83 ul
- Aliquot reaction mix 7.53 ul with multichannel pipette and mix well
- Put mineral oil in basin ~3 mL)
- Layer the capture reaction with 20ul mineral oil with multichannel pipette.
- Seal the plate with microseal and make sure that every well is completely sealed to prevent evaporation
- Centrifuge the capture plate for 3 min and place the plate on PCR machine with the following profile,
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_07_29) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_07_30)
SLN mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepared volume 220ul |
AmpliTaq Stoffel | 10.00 | U/ul | 2.00 | U/ul | 44.00 |
AmpLigase | 5.00 | U/ul | 0.50 | U/ul | 22.00 |
dNTP | 1000.00 | uM | 100.00 | uM | 22.00 |
10x AmpLigase Buffer | 10.00 | x | 1.00 | x | 22.00 |
H2O | 110.00 | ||||
Total | 220.00 |
- SLN mix
- Mix SLN mix in 1.5 mL tube
- Aliquot 27.5 of SLN mix into striptube
- Add 2 ul of SLN mix to each well with multi chennel pipette
- Exo I/III mix: for 110 rxn mix (prepare ~ more rxn extra)
- Mix 110ul each of ExoI and ExoIII in PCR tube and aliquot 27.5 ul into striptube
- Add 2ul of ExoI/III to each capture reaction with multichannel pipette
- Strictly suggestion: check the plate sealing every time to prevent sample evaporation
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX)[edit]
- Spin the capture plate for 3 min to collect all liquid to the bottom of the well
- Use atleast 2/3 of the total volume for amplification (total volume =25ul +2+2 = 29ul).
- I alway use 8-10 ul in each PCR reaction by trying out the first PCR to see the qPCR curve and the number of cycle then perform the second PCR
Components | 1rxn | 100 rxn mix |
Captured template | 10.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 200.00 |
10uM AmpR6.3 (IndX, X 1,2,3,…,96) | 2.00 | 0.00 |
50x SYBG | 0.80 | 80.00 |
2x Phusion MM | 50.00 | 5000.00 |
H2O | 35.20 | 3520.00 |
Total | 100.00 | Aliquot 88ul, add 10ul template and 2 ul of AmpR6.3 IndX |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
- Record the Ct value to compare the amplification of each sample
- The PCR can be stopped earlier depends on when it almost reach the plateau (do not over amplify).
Plate layout
A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 | A11 | A12 |
AmpR6.3 Ind1 | AmpR6.3 Ind9 | AmpR6.3 Ind17 | AmpR6.3 Ind25 | AmpR6.3 Ind33 | AmpR6.3 Ind41 | AmpR6.3 Ind49 | AmpR6.3 Ind57 | AmpR6.3 Ind65 | AmpR6.3 Ind73 | AmpR6.3 Ind81 | AmpR6.3 Ind89 |
B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | B11 | B12 |
AmpR6.3 Ind2 | AmpR6.3 Ind10 | AmpR6.3 Ind18 | AmpR6.3 Ind26 | AmpR6.3 Ind34 | AmpR6.3 Ind42 | AmpR6.3 Ind50 | AmpR6.3 Ind58 | AmpR6.3 Ind66 | AmpR6.3 Ind74 | AmpR6.3 Ind82 | AmpR6.3 Ind90 |
C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | C11 | C12 |
AmpR6.3 Ind3 | AmpR6.3 Ind11 | AmpR6.3 Ind19 | AmpR6.3 Ind27 | AmpR6.3 Ind35 | AmpR6.3 Ind43 | AmpR6.3 Ind51 | AmpR6.3 Ind59 | AmpR6.3 Ind67 | AmpR6.3 Ind75 | AmpR6.3 Ind83 | AmpR6.3 Ind91 |
D1 | D2 | D3 | D4 | D5 | D6 | D7 | D8 | D9 | D10 | D11 | D12 |
AmpR6.3 Ind4 | AmpR6.3 Ind12 | AmpR6.3 Ind20 | AmpR6.3 Ind28 | AmpR6.3 Ind36 | AmpR6.3 Ind44 | AmpR6.3 Ind52 | AmpR6.3 Ind60 | AmpR6.3 Ind68 | AmpR6.3 Ind76 | AmpR6.3 Ind84 | AmpR6.3 Ind92 |
E1 | E2 | E3 | E4 | E5 | E6 | E7 | E8 | E9 | E10 | E11 | E12 |
AmpR6.3 Ind5 | AmpR6.3 Ind13 | AmpR6.3 Ind21 | AmpR6.3 Ind29 | AmpR6.3 Ind37 | AmpR6.3 Ind45 | AmpR6.3 Ind53 | AmpR6.3 Ind61 | AmpR6.3 Ind69 | AmpR6.3 Ind77 | AmpR6.3 Ind85 | AmpR6.3 Ind93 |
F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 | F10 | F11 | F12 |
AmpR6.3 Ind6 | AmpR6.3 Ind14 | AmpR6.3 Ind22 | AmpR6.3 Ind30 | AmpR6.3 Ind38 | AmpR6.3 Ind46 | AmpR6.3 Ind54 | AmpR6.3 Ind62 | AmpR6.3 Ind70 | AmpR6.3 Ind78 | AmpR6.3 Ind86 | AmpR6.3 Ind94 |
G1 | G2 | G3 | G4 | G5 | G6 | G7 | G8 | G9 | G10 | G11 | G12 |
AmpR6.3 Ind7 | AmpR6.3 Ind15 | AmpR6.3 Ind23 | AmpR6.3 Ind31 | AmpR6.3 Ind39 | AmpR6.3 Ind47 | AmpR6.3 Ind55 | AmpR6.3 Ind63 | AmpR6.3 Ind71 | AmpR6.3 Ind79 | AmpR6.3 Ind87 | AmpR6.3 Ind95 |
H1 | H2 | H3 | H4 | H5 | H6 | H7 | H8 | H9 | H10 | H11 | H12 |
AmpR6.3 Ind8 | AmpR6.3 Ind16 | AmpR6.3 Ind24 | AmpR6.3 Ind32 | AmpR6.3 Ind40 | AmpR6.3 Ind48 | AmpR6.3 Ind56 | AmpR6.3 Ind64 | AmpR6.3 Ind72 | AmpR6.3 Ind80 | AmpR6.3 Ind88 | AmpR6.3 Ind96 |
- Note: I failed to amplify the captured DNA since most of the samples didn't show the amplification, and the PCR reaction set up was fine. I repeated PCR again for few samples to make sure that there was nothing wrong with the reaction set up. I still got the same result, so one possibility of this should be the DNA capture. At this time, I was not sure what wrong with the capture set up. However, I was very sure that I did every step right since Ling and Li helped me to check in every single step. I suspected about the reagents and probe S5 since it was just prepared while the other probes were the same. For the reagents, I used multiple tubes of ExoI/ExoIII from the old box and the expired dated was not shown. Another possibility, may be the AmpLigase enzyme and buffer since if the ligation didn't work, the circularization could not occur and it should be the reason rather than Exonuclease.
The steps below were not performed
- To purify the seq lib, 80ul of amplified amplicons were combined from two rounds (total volume of amplified amplicon = 160ul) into 96-well plate
- Purify seq lib with 0.8 volume Ampure beads, elute with 40ul EB
- Quantify DNA concentration of amplified amplicon in 6% TBE PAGE (12-well gel) (load 0.5ul of amplified amplicon)
- Pool ~25 ng of each amplified sampled into the same tube (=96X25 ng = 2.4ug)
- Perform PAGE size selection in 6% TBE PAGE (use ~ 4 of 1mm 5-well gel to prevent overloading of DNA )
- Purify DNA from acrylamide gel as previously described
- After precipitation and resuspen sequencing DNA with H2O ~ 40-50 ul, quantify DNA conc. by either PAGE quantification and Qubit dsDNA assay.
- Used the concentration from Qubit as the reference and the sequencing libraries are ready to be sequenced by HiSeq.