Noi/NOTES/2011-8-9
Jump to navigation
Jump to search
Repeat BSPP DMR330k capture of GA and control gDNA from blood sample
- 2011_08_08: received new plate of GA sample plate1 (so called GA plate1-2) from Ling. She prepared gDNA concentration 100ng/ul and volume ~10-15ul)
- However, after measured the conc. by Nanodrop the conc. of the samples were very varied and were not enough for bisulfite conversion. The concentrations in plate 1-1 were verified by Nanodrop again. Then I calculated the volume required for 1200ng. I didn't pool the gDNA from the two plates because they have different conc. and some sample has too low conc.
Bisulfite conversion
- Used EZ-96 DNA Methylation-Gold™ Kit (deep well format) Cat#D5008
- Eluted volume 20ul with Elution buffer. After Qubit ss-DNA assay the total yields will be calculated based on ~16ul left in the well.
Sample ID | Conc. (ng/ul) | Volume plate1-2 enough? | Volume plate1-2 (ul) | Amount plate1-2 | Conc. plate#1-1 (ng/ul) | Volume plate 1-1 | Sample ID | H2O (ul) | Total volume (ul) | CT conversion reagent (ul) | Total volume (ul) |
A1 | 177.30 | 6.77 | 1200.00 | A1 | 13.23 | 20.00 | 130.00 | 150.00 | |||
B1 | 104.91 | 11.44 | 1200.00 | B1 | 8.56 | 20.00 | 130.00 | 150.00 | |||
C1 | 108.20 | 11.09 | 1200.00 | C1 | 8.91 | 20.00 | 130.00 | 150.00 | |||
D1 | 95.30 | 12.59 | 1200.00 | D1 | 7.41 | 20.00 | 130.00 | 150.00 | |||
E1 | 142.71 | 8.41 | 1200.00 | E1 | 11.59 | 20.00 | 130.00 | 150.00 | |||
F1 | 130.28 | 9.21 | 1200.00 | F1 | 10.79 | 20.00 | 130.00 | 150.00 | |||
G1 | 195.56 | 6.14 | 1200.00 | G1 | 13.86 | 20.00 | 130.00 | 150.00 | |||
H1 | 126.34 | 9.50 | 1200.00 | H1 | 10.50 | 20.00 | 130.00 | 150.00 | |||
A2 | 119.33 | 10.06 | 1200.00 | A2 | 9.94 | 20.00 | 130.00 | 150.00 | |||
B2 | 135.56 | 8.85 | 1200.00 | B2 | 11.15 | 20.00 | 130.00 | 150.00 | |||
C2 | 48.65 | no | 10.00 | 486.50 | 94.48 | 7.55 | C2 | 2.45 | 20.00 | 130.00 | 150.00 |
D2 | 242.84 | 4.94 | 1200.00 | D2 | 15.06 | 20.00 | 130.00 | 150.00 | |||
E2 | 109.93 | 10.92 | 1200.00 | E2 | 9.08 | 20.00 | 130.00 | 150.00 | |||
F2 | 139.81 | 8.58 | 1200.00 | F2 | 11.42 | 20.00 | 130.00 | 150.00 | |||
G2 | 87.91 | 13.65 | 1200.00 | G2 | 6.35 | 20.00 | 130.00 | 150.00 | |||
H2 | 64.74 | no | 15.00 | 971.10 | 138.55 | 1.65 | H2 | 3.35 | 20.00 | 130.00 | 150.00 |
A3 | 121.07 | 9.91 | 1200.00 | A3 | 10.09 | 20.00 | 130.00 | 150.00 | |||
B3 | 103.64 | 11.58 | 1200.00 | B3 | 8.42 | 20.00 | 130.00 | 150.00 | |||
C3 | 76.09 | no | 12.00 | 913.08 | 144.84 | 1.98 | C3 | 6.02 | 20.00 | 130.00 | 150.00 |
D3 | 154.51 | 7.77 | 1200.00 | D3 | 12.23 | 20.00 | 130.00 | 150.00 | |||
E3 | 198.30 | 6.05 | 1200.00 | E3 | 13.95 | 20.00 | 130.00 | 150.00 | |||
F3 | 138.66 | 8.65 | 1200.00 | F3 | 11.35 | 20.00 | 130.00 | 150.00 | |||
G3 | 163.20 | 7.35 | 1200.00 | G3 | 12.65 | 20.00 | 130.00 | 150.00 | |||
H3 | 56.20 | no | 10.00 | 562.00 | 124.85 | 5.11 | H3 | 4.89 | 20.00 | 130.00 | 150.00 |
A4 | 98.78 | 12.15 | 1200.00 | A4 | 7.85 | 20.00 | 130.00 | 150.00 | |||
B4 | 60.47 | no | 12.00 | 725.64 | 151.88 | 3.12 | B4 | 4.88 | 20.00 | 130.00 | 150.00 |
C4 | 66.43 | no | 12.00 | 797.16 | 119.93 | 3.36 | C4 | 4.64 | 20.00 | 130.00 | 150.00 |
D4 | 119.64 | 10.03 | 1200.00 | D4 | 9.97 | 20.00 | 130.00 | 150.00 | |||
E4 | 208.77 | 5.75 | 1200.00 | E4 | 14.25 | 20.00 | 130.00 | 150.00 | |||
F4 | 83.48 | no | 12.00 | 1001.76 | 58.64 | 3.38 | F4 | 4.62 | 20.00 | 130.00 | 150.00 |
G4 | 71.01 | no | 12.00 | 852.12 | 36.48 | 9.54 | G4 | 0.00 | 21.54 | 130.00 | 151.54 |
H4 | 210.32 | 5.71 | 1200.00 | H4 | 14.29 | 20.00 | 130.00 | 150.00 | |||
A5 | 266.74 | 4.50 | 1200.00 | A5 | 15.50 | 20.00 | 130.00 | 150.00 | |||
B5 | 105.58 | 11.37 | 1200.00 | B5 | 8.63 | 20.00 | 130.00 | 150.00 | |||
C5 | 89.41 | 13.42 | 1200.00 | C5 | 6.58 | 20.00 | 130.00 | 150.00 | |||
D5 | 61.84 | no | 15.00 | 927.60 | 177.45 | 1.54 | D5 | 3.46 | 20.00 | 130.00 | 150.00 |
E5 | 66.71 | no | 12.00 | 800.52 | 135.97 | 2.94 | E5 | 5.06 | 20.00 | 130.00 | 150.00 |
F5 | 125.29 | 9.58 | 1200.00 | F5 | 10.42 | 20.00 | 130.00 | 150.00 | |||
G5 | 121.31 | 9.89 | 1200.00 | G5 | 10.11 | 20.00 | 130.00 | 150.00 | |||
H5 | 88.27 | 13.59 | 1200.00 | H5 | 6.41 | 20.00 | 130.00 | 150.00 | |||
A6 | 134.70 | 8.91 | 1200.00 | A6 | 11.09 | 20.00 | 130.00 | 150.00 | |||
B6 | 62.68 | no | 12.00 | 752.16 | 154.46 | 2.90 | B6 | 5.10 | 20.00 | 130.00 | 150.00 |
C6 | 141.72 | 8.47 | 1200.00 | C6 | 11.53 | 20.00 | 130.00 | 150.00 | |||
D6 | 187.04 | 6.42 | 1200.00 | D6 | 13.58 | 20.00 | 130.00 | 150.00 | |||
E6 | 64.02 | no | 12.00 | 768.24 | 138.92 | 3.11 | E6 | 4.89 | 20.00 | 130.00 | 150.00 |
F6 | 58.78 | no | 12.00 | 705.36 | 188.15 | 2.63 | F6 | 5.37 | 20.00 | 130.00 | 150.00 |
G6 | 170.00 | 7.06 | 1200.00 | G6 | 12.94 | 20.00 | 130.00 | 150.00 | |||
H6 | 111.00 | 10.81 | 1200.00 | H6 | 9.19 | 20.00 | 130.00 | 150.00 | |||
A7 | 320.17 | 3.75 | 1200.00 | A7 | 16.25 | 20.00 | 130.00 | 150.00 | |||
B7 | 162.36 | 7.39 | 1200.00 | B7 | 12.61 | 20.00 | 130.00 | 150.00 | |||
C7 | 126.32 | 9.50 | 1200.00 | C7 | 10.50 | 20.00 | 130.00 | 150.00 | |||
D7 | 368.67 | 3.25 | 1200.00 | D7 | 16.75 | 20.00 | 130.00 | 150.00 | |||
E7 | 117.82 | 10.19 | 1200.00 | E7 | 9.81 | 20.00 | 130.00 | 150.00 | |||
F7 | 84.38 | no | 12.00 | 1012.56 | 107.02 | 1.75 | F7 | 6.25 | 20.00 | 130.00 | 150.00 |
G7 | 105.04 | 11.42 | 1200.00 | G7 | 8.58 | 20.00 | 130.00 | 150.00 | |||
H7 | 86.61 | 13.86 | 1200.00 | H7 | 6.14 | 20.00 | 130.00 | 150.00 | |||
A8 | 95.35 | 12.59 | 1200.00 | A8 | 7.41 | 20.00 | 130.00 | 150.00 | |||
B8 | 98.32 | 12.21 | 1200.00 | B8 | 7.79 | 20.00 | 130.00 | 150.00 | |||
C8 | 129.29 | 9.28 | 1200.00 | C8 | 10.72 | 20.00 | 130.00 | 150.00 | |||
D8 | 100.98 | 11.88 | 1200.00 | D8 | 8.12 | 20.00 | 130.00 | 150.00 | |||
E8 | 73.13 | no | 12.00 | 877.56 | 132.82 | 2.43 | E8 | 5.57 | 20.00 | 130.00 | 150.00 |
F8 | 111.10 | 10.80 | 1200.00 | F8 | 9.20 | 20.00 | 130.00 | 150.00 | |||
G8 | 30.00 | no | 10.00 | 300.00 | 162.10 | 5.55 | G8 | 4.45 | 20.00 | 130.00 | 150.00 |
H8 | 82.39 | no | 12.00 | 988.68 | 182.77 | 1.16 | H8 | 6.84 | 20.00 | 130.00 | 150.00 |
A9 | 61.85 | no | 12.00 | 742.20 | 136.13 | 3.36 | A9 | 4.64 | 20.00 | 130.00 | 150.00 |
B9 | 110.49 | 10.86 | 1200.00 | B9 | 9.14 | 20.00 | 130.00 | 150.00 | |||
C9 | 62.30 | no | 12.00 | 747.60 | 123.23 | 3.67 | C9 | 4.33 | 20.00 | 130.00 | 150.00 |
D9 | 118.40 | 10.14 | 1200.00 | D9 | 9.86 | 20.00 | 130.00 | 150.00 | |||
E9 | 75.01 | 16.00 | 1200.00 | E9 | 4.00 | 20.00 | 130.00 | 150.00 | |||
F9 (tube 49.9) | 60.63 | no | 15.00 | 909.45 | 38.24 | 7.60 | F9 (tube 49.9) | 0.00 | 22.60 | 130.00 | 152.60 |
G9 | 91.20 | no | 12.00 | 1094.40 | 142.40 | 0.74 | G9 | 7.26 | 20.00 | 130.00 | 150.00 |
H9 | 90.62 | 13.24 | 1200.00 | H9 | 6.76 | 20.00 | 130.00 | 150.00 | |||
A10 | 83.92 | no | 13.00 | 1090.96 | 193.87 | 0.56 | A10 | 6.44 | 20.00 | 130.00 | 150.00 |
B10 | 120.56 | 9.95 | 1200.00 | B10 | 10.05 | 20.00 | 130.00 | 150.00 | |||
C10 | 86.38 | no | 12.00 | 1036.56 | 168.48 | 0.97 | C10 | 7.03 | 20.00 | 130.00 | 150.00 |
D10 | 49.85 | no | 12.00 | 598.20 | 172.60 | 3.49 | D10 | 4.51 | 20.00 | 130.00 | 150.00 |
E10 | 123.63 | 9.71 | 1200.00 | E10 | 10.29 | 20.00 | 130.00 | 150.00 | |||
F10 | 14.38 | no | 12.00 | 172.56 | 89.90 | 11.43 | F10 | 0.00 | 23.43 | 130.00 | 153.43 |
G10 | 60.43 | no | 12.00 | 725.16 | 163.82 | 2.90 | G10 | 5.10 | 20.00 | 130.00 | 150.00 |
H10 | 71.01 | no | 12.00 | 852.12 | 251.70 | 1.38 | H10 | 6.62 | 20.00 | 130.00 | 150.00 |
A11 | 71.94 | no | 12.00 | 863.28 | 96.54 | 3.49 | A11 | 4.51 | 20.00 | 130.00 | 150.00 |
B11 | 72.80 | no | 12.00 | 873.60 | 181.22 | 1.80 | B11 | 6.20 | 20.00 | 130.00 | 150.00 |
C11 | 43.85 | no | 12.00 | 526.20 | 192.17 | 3.51 | C11 | 4.49 | 20.00 | 130.00 | 150.00 |
D11 | 201.88 | 5.94 | 1200.00 | D11 | 14.06 | 20.00 | 130.00 | 150.00 | |||
E11 | 221.63 | 5.41 | 1200.00 | E11 | 14.59 | 20.00 | 130.00 | 150.00 | |||
F11 | 68.94 | no | 12.00 | 827.28 | 139.05 | 2.68 | F11 | 5.32 | 20.00 | 130.00 | 150.00 |
G11 | 76.28 | no | 12.00 | 915.36 | 137.85 | 2.06 | G11 | 5.94 | 20.00 | 130.00 | 150.00 |
H11 | 73.95 | no | 12.00 | 887.40 | 126.45 | 2.47 | H11 | 5.53 | 20.00 | 130.00 | 150.00 |
A12 | 88.25 | no | 12.00 | 1059.00 | 126.33 | 1.12 | A12 | 6.88 | 20.00 | 130.00 | 150.00 |
B12 (tube 94.9) | 109.45 | 10.96 | 1200.00 | B12 (tube 94.9) | 9.04 | 20.00 | 130.00 | 150.00 | |||
C12 | 59.30 | no | 12.00 | 711.60 | 147.66 | 3.31 | C12 | 4.69 | 20.00 | 130.00 | 150.00 |
D12 | 42.39 | no | 12.00 | 508.68 | 153.07 | 4.52 | D12 | 3.48 | 20.00 | 130.00 | 150.00 |
E12 | 63.68 | no | 12.00 | 764.16 | 138.06 | 3.16 | E12 | 4.84 | 20.00 | 130.00 | 150.00 |
F12 | 26.25 | no | 12.00 | 315.00 | 144.07 | 6.14 | F12 | 1.86 | 20.00 | 130.00 | 150.00 |
G12 | 59.01 | no | 12.00 | 708.12 | 123.67 | 3.98 | G12 | 4.02 | 20.00 | 130.00 | 150.00 |
H12 | 142.64 | 8.41 | 1200.00 | H12 | 11.59 | 20.00 | 130.00 | 150.00 |
Qubit ssDNA assay
- Follow protocol provided with the kit
- Took 1ul of bis-cvt DNA into 9ul of H2O, then add the diluted DNA to 190ul ssDN Qubit reagent (dilution factor = 200)
Order | Well ID | Conc. in Qubit (ng/mL) | Volume (ul) | Final conc. (ng/ul) | Yields (ng) |
1.00 | A1 | 217.00 | 16.00 | 43.40 | 694.40 |
2.00 | B1 | 180.00 | 16.00 | 36.00 | 576.00 |
3.00 | C1 | 204.00 | 16.00 | 40.80 | 652.80 |
4.00 | D1 | 211.00 | 16.00 | 42.20 | 675.20 |
5.00 | E1 | 201.00 | 16.00 | 40.20 | 643.20 |
6.00 | F1 | 150.00 | 16.00 | 30.00 | 480.00 |
7.00 | G1 | 135.00 | 16.00 | 27.00 | 432.00 |
8.00 | H1 | 219.00 | 16.00 | 43.80 | 700.80 |
9.00 | A2 | 190.00 | 16.00 | 38.00 | 608.00 |
10.00 | B2 | 170.00 | 16.00 | 34.00 | 544.00 |
11.00 | C2 | 151.00 | 16.00 | 30.20 | 483.20 |
12.00 | D2 | 153.00 | 16.00 | 30.60 | 489.60 |
13.00 | E2 | 292.00 | 16.00 | 58.40 | 934.40 |
14.00 | F2 | 112.00 | 16.00 | 22.40 | 358.40 |
15.00 | G2 | 160.00 | 16.00 | 32.00 | 512.00 |
16.00 | H2 | 105.00 | 16.00 | 21.00 | 336.00 |
17.00 | A3 | 176.00 | 16.00 | 35.20 | 563.20 |
18.00 | B3 | 137.00 | 16.00 | 27.40 | 438.40 |
19.00 | C3 | 119.00 | 16.00 | 23.80 | 380.80 |
20.00 | D3 | 128.00 | 16.00 | 25.60 | 409.60 |
21.00 | E3* | 40.20 | 16.00 | 8.04 | 128.64 |
22.00 | F3 | 134.00 | 16.00 | 26.80 | 428.80 |
23.00 | G3 | 176.00 | 16.00 | 35.20 | 563.20 |
24.00 | H3 | 112.00 | 16.00 | 22.40 | 358.40 |
25.00 | A4 | 144.00 | 16.00 | 28.80 | 460.80 |
26.00 | B4 | 135.00 | 16.00 | 27.00 | 432.00 |
27.00 | C4 | 133.00 | 16.00 | 26.60 | 425.60 |
28.00 | D4 | 139.00 | 16.00 | 27.80 | 444.80 |
29.00 | E4 | 133.00 | 16.00 | 26.60 | 425.60 |
30.00 | F4 | 189.00 | 16.00 | 37.80 | 604.80 |
31.00 | G4 | 88.60 | 16.00 | 17.72 | 283.52 |
32.00 | H4* | 47.10 | 16.00 | 9.42 | 150.72 |
33.00 | A5 | 148.00 | 16.00 | 29.60 | 473.60 |
34.00 | B5 | 127.00 | 16.00 | 25.40 | 406.40 |
35.00 | C5 | 83.70 | 16.00 | 16.74 | 267.84 |
36.00 | D5 | 120.00 | 16.00 | 24.00 | 384.00 |
37.00 | E5 | 117.00 | 16.00 | 23.40 | 374.40 |
38.00 | F5* | 34.40 | 16.00 | 6.88 | 110.08 |
39.00 | G5 | 104.00 | 16.00 | 20.80 | 332.80 |
40.00 | H5 | 112.00 | 16.00 | 22.40 | 358.40 |
41.00 | A6 | 125.00 | 16.00 | 25.00 | 400.00 |
42.00 | B6 | 126.00 | 16.00 | 25.20 | 403.20 |
43.00 | C6 | 149.00 | 16.00 | 29.80 | 476.80 |
44.00 | D6 | 135.00 | 16.00 | 27.00 | 432.00 |
45.00 | E6 | 110.00 | 16.00 | 22.00 | 352.00 |
46.00 | F6 | 102.00 | 16.00 | 20.40 | 326.40 |
47.00 | G6 | 127.00 | 16.00 | 25.40 | 406.40 |
48.00 | H6 | 59.40 | 16.00 | 11.88 | 190.08 |
49.00 | A7 | 161.00 | 16.00 | 32.20 | 515.20 |
50.00 | B7 | 146.00 | 16.00 | 29.20 | 467.20 |
51.00 | C7 | 160.00 | 16.00 | 32.00 | 512.00 |
52.00 | D7 | 135.00 | 16.00 | 27.00 | 432.00 |
53.00 | E7 | 146.00 | 16.00 | 29.20 | 467.20 |
54.00 | F7 | 133.00 | 16.00 | 26.60 | 425.60 |
55.00 | G7 | 143.00 | 16.00 | 28.60 | 457.60 |
56.00 | H7 | 85.20 | 16.00 | 17.04 | 272.64 |
57.00 | A8 | 141.00 | 16.00 | 28.20 | 451.20 |
58.00 | B8 | 147.00 | 16.00 | 29.40 | 470.40 |
59.00 | C8 | 119.00 | 16.00 | 23.80 | 380.80 |
60.00 | D8 | 81.40 | 16.00 | 16.28 | 260.48 |
61.00 | E8 | 120.00 | 16.00 | 24.00 | 384.00 |
62.00 | F8 | 62.20 | 16.00 | 12.44 | 199.04 |
63.00 | G8 | 106.00 | 16.00 | 21.20 | 339.20 |
64.00 | H8 | 135.00 | 16.00 | 27.00 | 432.00 |
65.00 | A9 | 110.00 | 16.00 | 22.00 | 352.00 |
66.00 | B9 | 147.00 | 16.00 | 29.40 | 470.40 |
67.00 | C9 | 127.00 | 16.00 | 25.40 | 406.40 |
68.00 | D9 | 95.70 | 16.00 | 19.14 | 306.24 |
69.00 | E9 | 99.80 | 16.00 | 19.96 | 319.36 |
70.00 | F9 | 104.00 | 16.00 | 20.80 | 332.80 |
71.00 | G9 | 92.50 | 16.00 | 18.50 | 296.00 |
72.00 | H9 | 116.00 | 16.00 | 23.20 | 371.20 |
73.00 | A10 | 103.00 | 16.00 | 20.60 | 329.60 |
74.00 | B10 | 69.70 | 16.00 | 13.94 | 223.04 |
75.00 | C10 | 121.00 | 16.00 | 24.20 | 387.20 |
76.00 | D10 | 141.00 | 16.00 | 28.20 | 451.20 |
77.00 | E10 | 114.00 | 16.00 | 22.80 | 364.80 |
78.00 | F10 | 154.00 | 16.00 | 30.80 | 492.80 |
79.00 | G10 | 124.00 | 16.00 | 24.80 | 396.80 |
80.00 | H10 | 111.00 | 16.00 | 22.20 | 355.20 |
81.00 | A11 | 82.20 | 16.00 | 16.44 | 263.04 |
82.00 | B11 | 112.00 | 16.00 | 22.40 | 358.40 |
83.00 | C11 | 109.00 | 16.00 | 21.80 | 348.80 |
84.00 | D11 | 61.50 | 16.00 | 12.30 | 196.80 |
85.00 | E11 | 65.20 | 16.00 | 13.04 | 208.64 |
86.00 | F11 | 148.00 | 16.00 | 29.60 | 473.60 |
87.00 | G11 | 133.00 | 16.00 | 26.60 | 425.60 |
88.00 | H11 | 200.00 | 16.00 | 40.00 | 640.00 |
89.00 | A12 | 114.00 | 16.00 | 22.80 | 364.80 |
90.00 | B12 | 149.00 | 16.00 | 29.80 | 476.80 |
91.00 | C12 | 164.00 | 16.00 | 32.80 | 524.80 |
92.00 | D12 | 142.00 | 16.00 | 28.40 | 454.40 |
93.00 | E12 | 145.00 | 16.00 | 29.00 | 464.00 |
94.00 | F12 | 137.00 | 16.00 | 27.40 | 438.40 |
95.00 | G12 | 177.00 | 16.00 | 35.40 | 566.40 |
96.00 | H12 | 202.00 | 16.00 | 40.40 | 646.40 |
- Note: * = samples which total yields < 200ng
Capture set up
Probe calculation
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 | 31.20 | 2.93 | 307.22 |
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 |
- check volume of every well if they have the same level
Reaction mix
Components | 1rxn (ul) | 105 rxn mix (ul) |
10X AmpLigase Buffer | 2.50 | 262.50 |
DMR330k probe mix (ul) | 4.53 | 475.65 |
105nM Oligo suppressor | 0.30 | 31.50 |
Total volume (ul) | 7.33 | 769.65 |
sample ID | Final conc. (ng/ul) | Volume for 300ng(ul) | sample ID | 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 | 43.40 | 6.91 | A1 | 10.76 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B1 | 36.00 | 8.33 | B1 | 9.34 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C1 | 40.80 | 7.35 | C1 | 10.32 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D1 | 42.20 | 7.11 | D1 | 10.56 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E1 | 40.20 | 7.46 | E1 | 10.21 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F1 | 30.00 | 10.00 | F1 | 7.67 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G1 | 27.00 | 11.11 | G1 | 6.56 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H1 | 43.80 | 6.85 | H1 | 10.82 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A2 | 38.00 | 7.89 | A2 | 9.78 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B2 | 34.00 | 8.82 | B2 | 8.85 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C2 | 30.20 | 9.93 | C2 | 7.74 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D2 | 30.60 | 9.80 | D2 | 7.87 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E2 | 58.40 | 5.14 | E2 | 12.53 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F2 | 22.40 | 13.39 | F2 | 4.28 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G2 | 32.00 | 9.38 | G2 | 8.30 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H2 | 21.00 | 14.29 | H2 | 3.38 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A3 | 35.20 | 8.52 | A3 | 9.15 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B3 | 27.40 | 10.95 | B3 | 6.72 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C3 | 23.80 | 12.61 | C3 | 5.06 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D3 | 25.60 | 11.72 | D3 | 5.95 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E3* | 8.04 | 16.00 | E3* | 1.67 | 128.64 | 4.53 | 2.50 | 0.30 | 25.00 |
F3 | 26.80 | 11.19 | F3 | 6.48 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G3 | 35.20 | 8.52 | G3 | 9.15 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H3 | 22.40 | 13.39 | H3 | 4.28 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A4 | 28.80 | 10.42 | A4 | 7.25 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B4 | 27.00 | 11.11 | B4 | 6.56 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C4 | 26.60 | 11.28 | C4 | 6.39 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D4 | 27.80 | 10.79 | D4 | 6.88 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E4 | 26.60 | 11.28 | E4 | 6.39 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F4 | 37.80 | 7.94 | F4 | 9.73 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G4 | 17.72 | 16.00 | G4 | 1.67 | 283.52 | 4.53 | 2.50 | 0.30 | 25.00 |
H4* | 9.42 | 16.00 | H4* | 1.67 | 150.72 | 4.53 | 2.50 | 0.30 | 25.00 |
A5 | 29.60 | 10.14 | A5 | 7.53 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B5 | 25.40 | 11.81 | B5 | 5.86 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C5 | 16.74 | 16.00 | C5 | 1.67 | 267.84 | 4.53 | 2.50 | 0.30 | 25.00 |
D5 | 24.00 | 12.50 | D5 | 5.17 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E5 | 23.40 | 12.82 | E5 | 4.85 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F5* | 6.88 | 16.00 | F5* | 1.67 | 110.08 | 4.53 | 2.50 | 0.30 | 25.00 |
G5 | 20.80 | 14.42 | G5 | 3.25 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H5 | 22.40 | 13.39 | H5 | 4.28 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A6 | 25.00 | 12.00 | A6 | 5.67 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B6 | 25.20 | 11.90 | B6 | 5.77 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C6 | 29.80 | 10.07 | C6 | 7.60 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D6 | 27.00 | 11.11 | D6 | 6.56 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E6 | 22.00 | 13.64 | E6 | 4.03 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F6 | 20.40 | 14.71 | F6 | 2.96 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G6 | 25.40 | 11.81 | G6 | 5.86 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H6 | 11.88 | 16.00 | H6 | 1.67 | 190.08 | 4.53 | 2.50 | 0.30 | 25.00 |
A7 | 32.20 | 9.32 | A7 | 8.35 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B7 | 29.20 | 10.27 | B7 | 7.40 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C7 | 32.00 | 9.38 | C7 | 8.30 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D7 | 27.00 | 11.11 | D7 | 6.56 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E7 | 29.20 | 10.27 | E7 | 7.40 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F7 | 26.60 | 11.28 | F7 | 6.39 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G7 | 28.60 | 10.49 | G7 | 7.18 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H7 | 17.04 | 16.00 | H7 | 1.67 | 272.64 | 4.53 | 2.50 | 0.30 | 25.00 |
A8 | 28.20 | 10.64 | A8 | 7.03 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B8 | 29.40 | 10.20 | B8 | 7.47 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C8 | 23.80 | 12.61 | C8 | 5.06 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D8 | 16.28 | 16.00 | D8 | 1.67 | 260.48 | 4.53 | 2.50 | 0.30 | 25.00 |
E8 | 24.00 | 12.50 | E8 | 5.17 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F8 | 12.44 | 16.00 | F8 | 1.67 | 199.04 | 4.53 | 2.50 | 0.30 | 25.00 |
G8 | 21.20 | 14.15 | G8 | 3.52 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H8 | 27.00 | 11.11 | H8 | 6.56 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A9 | 22.00 | 13.64 | A9 | 4.03 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B9 | 29.40 | 10.20 | B9 | 7.47 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C9 | 25.40 | 11.81 | C9 | 5.86 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D9 | 19.14 | 15.67 | D9 | 2.00 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E9 | 19.96 | 15.03 | E9 | 2.64 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F9 | 20.80 | 14.42 | F9 | 3.25 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G9 | 18.50 | 16.00 | G9 | 1.67 | 296.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H9 | 23.20 | 12.93 | H9 | 4.74 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A10 | 20.60 | 14.56 | A10 | 3.11 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B10 | 13.94 | 16.00 | B10 | 1.67 | 223.04 | 4.53 | 2.50 | 0.30 | 25.00 |
C10 | 24.20 | 12.40 | C10 | 5.27 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D10 | 28.20 | 10.64 | D10 | 7.03 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E10 | 22.80 | 13.16 | E10 | 4.51 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F10 | 30.80 | 9.74 | F10 | 7.93 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G10 | 24.80 | 12.10 | G10 | 5.57 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H10 | 22.20 | 13.51 | H10 | 4.16 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A11 | 16.44 | 16.00 | A11 | 1.67 | 263.04 | 4.53 | 2.50 | 0.30 | 25.00 |
B11 | 22.40 | 13.39 | B11 | 4.28 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C11 | 21.80 | 13.76 | C11 | 3.91 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D11 | 12.30 | 16.00 | D11 | 1.67 | 196.80 | 4.53 | 2.50 | 0.30 | 25.00 |
E11 | 13.04 | 16.00 | E11 | 1.67 | 208.64 | 4.53 | 2.50 | 0.30 | 25.00 |
F11 | 29.60 | 10.14 | F11 | 7.53 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G11 | 26.60 | 11.28 | G11 | 6.39 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H11 | 40.00 | 7.50 | H11 | 10.17 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
A12 | 22.80 | 13.16 | A12 | 4.51 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
B12 | 29.80 | 10.07 | B12 | 7.60 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
C12 | 32.80 | 9.15 | C12 | 8.52 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
D12 | 28.40 | 10.56 | D12 | 7.11 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
E12 | 29.00 | 10.34 | E12 | 7.33 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
F12 | 27.40 | 10.95 | F12 | 6.72 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
G12 | 35.40 | 8.47 | G12 | 9.20 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
H12 | 40.40 | 7.43 | H12 | 10.24 | 300.00 | 4.53 | 2.50 | 0.30 | 25.00 |
- 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): 96.21 ul
- Aliquot reaction mix 7.33 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
- Check the level of every well that they have the same level
- 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 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_08_14) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_15)
SLN mix
- Prepare 275 ul (2.5ul/rxn and prepare total for 110 rxn, more extra reaction to ensure that the volume enough when pipetting)
Components | Stock conc. | Unit | Final conc. | Unit | Prepared volume 275ul |
AmpliTaq Stoffel | 10.00 | U/ul | 2.00 | U/ul | 55.00 |
AmpLigase | 5.00 | U/ul | 0.50 | U/ul | 27.50 |
dNTP | 1000.00 | uM | 100.00 | uM | 27.50 |
10x AmpLigase Buffer | 10.00 | x | 1.00 | x | 27.50 |
H2O | 137.50 | ||||
Total | 275.00 |
- Aliquot 34.38 of SLN mix to strip tube and add 2.5ul to each well with multi-channel pipette
PAGE Quantification of Sequencing Libraries
Well ID | conc. (ng/ul) | Total amount in 60ul (ng) | Volume for 25ng (ul) | Well ID | conc. (ng/ul) | Total amount in 60ul (ng) | Volume for 25ng (ul) | Well ID | conc. (ng/ul) | Total amount in 60ul (ng) | Volume for 25ng (ul) |
A1 | 7.42 | 445.24 | 3.37 | C1 | 5.62 | 337.48 | 4.44 | E1 | 7.35 | 440.82 | 3.40 |
A2 | 5.64 | 338.19 | 4.44 | C2 | 4.60 | 276.17 | 5.43 | E2 | 6.74 | 404.16 | 3.71 |
A3 | 4.53 | 271.61 | 5.52 | C3 | 3.07 | 184.27 | 8.14 | E3** | 0.19 | 11.46 | 60** |
A4 | 4.10 | 245.97 | 6.10 | C4 | 2.64 | 158.13 | 9.49 | E4 | 3.41 | 204.74 | 7.33 |
A5 | 4.34 | 260.26 | 5.76 | C5 | 0.95 | 56.71 | 26.45 | E5 | 1.90 | 114.02 | 13.16 |
A6 | 4.37 | 262.10 | 5.72 | C6 | 4.85 | 290.89 | 5.16 | E6 | 1.36 | 81.30 | 18.45 |
A7 | 3.64 | 218.44 | 6.87 | C7 | 4.26 | 255.41 | 5.87 | E7 | 3.26 | 195.32 | 7.68 |
A8 | 9.62 | 577.08 | 2.60 | C8 | 2.28 | 136.64 | 10.98 | E8 | 2.49 | 149.17 | 10.06 |
A9 | 5.87 | 352.21 | 4.26 | C9 | 0.65 | 39.17 | 38.30 | E9 | 1.36 | 81.74 | 18.35 |
A10 | 7.38 | 442.57 | 3.39 | C10 | 2.01 | 120.65 | 12.43 | E10 | 2.20 | 132.04 | 11.36 |
B1 | 5.84 | 350.35 | 4.28 | D1 | 5.56 | 333.37 | 4.50 | F1 | 6.67 | 400.42 | 3.75 |
B2 | 5.46 | 327.84 | 4.58 | D2 | 4.53 | 271.74 | 5.52 | F2 | 3.02 | 181.16 | 8.28 |
B3 | 2.03 | 121.68 | 12.33 | D3 | 1.88 | 112.91 | 13.28 | F3 | 3.12 | 187.27 | 8.01 |
B4 | 2.88 | 172.65 | 8.69 | D4 | 2.64 | 158.53 | 9.46 | F4 | 2.70 | 162.27 | 9.24 |
B5 | 3.77 | 226.31 | 6.63 | D5 | 1.90 | 114.20 | 13.14 | F5** | 0.10 | 6.08 | 60** |
B6 | 3.43 | 205.99 | 7.28 | D6 | 2.82 | 169.46 | 8.85 | F6 | 2.18 | 130.98 | 11.45 |
B7 | 4.04 | 242.53 | 6.18 | D7 | 2.95 | 177.20 | 8.47 | F7 | 1.20 | 71.81 | 20.89 |
B8 | 3.92 | 235.42 | 6.37 | D8** | -0.45 | -26.73 | 60** | F8 | 0.73 | 43.78 | 34.26 |
B9 | 3.46 | 207.79 | 7.22 | D9 | 1.52 | 91.15 | 16.46 | F9 | 0.72 | 43.22 | 34.71 |
B10 | 0.58 | 34.94 | 42.93 | D10 | 3.01 | 180.35 | 8.32 | F10 | 4.79 | 287.50 | 5.22 |
Well ID | conc. (ng/ul) | Total amount in 60ul (ng) | Volume for 25ng (ul) | Well ID | conc. (ng/ul) | Total amount in 60ul (ng) | Volume for 25ng (ul) |
G1 | 5.54 | 332.34 | 4.51 | A11 | 2.96 | 177.41 | 8.45 |
G2 | 4.10 | 245.73 | 6.10 | B11 | 3.01 | 180.38 | 8.32 |
G3 | 3.70 | 222.02 | 6.76 | C11 | 3.71 | 222.85 | 6.73 |
G4 | 0.89 | 53.24 | 28.17 | D11 | 0.54 | 32.69 | 45.88 |
G5 | 1.61 | 96.85 | 15.49 | E11 | 1.33 | 80.08 | 18.73 |
G6 | 3.74 | 224.69 | 6.68 | F11 | 3.86 | 231.73 | 6.47 |
G7 | 2.78 | 166.71 | 9.00 | G11 | 3.33 | 199.56 | 7.52 |
G8 | 1.18 | 70.72 | 21.21 | H11 | 4.59 | 275.44 | 5.45 |
G9 | 0.80 | 48.13 | 31.16 | A12 | 6.58 | 394.93 | 3.80 |
G10 | 3.28 | 196.77 | 7.62 | B12 | 4.96 | 297.75 | 5.04 |
H1 | 5.58 | 334.76 | 4.48 | C12 | 5.13 | 307.60 | 4.88 |
H2 | 3.56 | 213.89 | 7.01 | D12 | 4.86 | 291.87 | 5.14 |
H3 | 4.43 | 266.06 | 5.64 | E12 | 5.25 | 314.85 | 4.76 |
H4 | 1.13 | 67.98 | 22.07 | F12 | 4.58 | 274.81 | 5.46 |
H5 | 2.94 | 176.59 | 8.49 | G12 | 6.25 | 375.00 | 4.00 |
H6 | 3.21 | 192.76 | 7.78 | H12 | 3.27 | 196.30 | 7.64 |
H7 | 1.73 | 103.53 | 14.49 | ||||
H8 | 6.92 | 415.47 | 3.61 | ||||
H9 | 2.34 | 140.57 | 10.67 | ||||
H10 | 6.04 | 362.62 | 4.14 |