Noi/NOTES/2011-8-14
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BSPP DMR330k capture of brain sample from Dr. Ophoff's lab (UCLA)
- August 1, 2011, received 96 samples from Dr. Ophoff's lab
- Tina Wang confirmed the concentration of each sample was about 50ng/ul
- The concentrations were determined again by Nanadrop, and it turned out that the concentrations were vere varied. The volume of the gDNA needed to be adjust when perform bisulfite conversion
- Note: The ratio of 260/230nm of most of the samples were very low, so I assumed high salt contamination.
Nanodrop measurement
Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul | Well ID | ng/ul |
A1 | 127.53 | A2 | 101.77 | A3 | 104.16 | A4 | 89.42 | A5 | 108.53 | A6 | 98.50 | A7 | 71.86 | A8 | 59.88 | A9 | 76.42 | A10 | 59.93 | A11 | 152.47 | A12 | 74.20 |
B1 | 214.10 | B2 | 109.05 | B3 | 128.33 | B4 | 80.22 | B5 | 108.85 | B6 | 74.95 | B7 | 39.82 | B8 | 85.67 | B9 | 228.10 | B10 | 72.25 | B11 | 90.51 | B12 | 92.20 |
C1 | 98.39 | C2 | 106.42 | C3 | 90.52 | C4 | 114.26 | C5 | 100.28 | C6 | 91.90 | C7 | 79.72 | C8 | 96.65 | C9 | 119.19 | C10 | 69.85 | C11 | 106.38 | C12 | 46.85 |
D1 | 102.29 | D2 | 94.38 | D3 | 75.38 | D4 | 119.02 | D5 | 109.51 | D6 | 63.31 | D7 | 100.77 | D8 | 128.37 | D9 | 269.45 | D10 | 129.33 | D11 | 88.70 | D12 | 68.39 |
E1 | 90.70 | E2 | 84.67 | E3 | 139.81 | E4 | 90.16 | E5 | 98.92 | E6 | 76.35 | E7 | 197.06 | E8 | 89.01 | E9 | 49.91 | E10 | 62.05 | E11 | 78.12 | E12 | 83.77 |
F1 | 55.57 | F2 | 108.48 | F3 | 67.75 | F4 | 117.73 | F5 | 114.30 | F6 | 77.34 | F7 | 60.37 | F8 | 95.33 | F9 | 86.00 | F10 | 139.09 | F11 | 61.46 | F12 | 53.64 |
G1 | 111.01 | G2 | 62.01 | G3 | 98.83 | G4 | 133.36 | G5 | 166.66 | G6 | 100.09 | G7 | 53.66 | G8 | 84.04 | G9 | 593.63 | G10 | 189.64 | G11 | 111.77 | G12 | 106.40 |
H1 | 108.95 | H2 | 57.54 | H3 | 83.68 | H4 | 90.89 | H5 | 100.31 | H6 | 76.76 | H7 | 55.88 | H8 | 144.05 | H9 | 51.54 | H10 | 70.48 | H11 | 125.43 | H12 | 74.57 |
Bisulfite conversion
- Used EZ-96 DNA Methylation-Gold™ Kit (deep well format) Cat#D5008
Sample ID | ng/ul | Volume for 1200ng | Sample ID | H2O | Total amount (ng) | CT conversion reagent (ul) | Total volume (ul) |
A1 | 127.53 | 9.41 | A1 | 10.59 | 1200.00 | 130.00 | 150.00 |
B1 | 214.10 | 5.60 | B1 | 14.40 | 1200.00 | 130.00 | 150.00 |
C1 | 98.39 | 12.20 | C1 | 7.80 | 1200.00 | 130.00 | 150.00 |
D1 | 102.29 | 11.73 | D1 | 8.27 | 1200.00 | 130.00 | 150.00 |
E1 | 90.70 | 13.23 | E1 | 6.77 | 1200.00 | 130.00 | 150.00 |
F1* | 55.57 | 20.00 | F1* | 0.00 | 1111.40 | 130.00 | 150.00 |
G1 | 111.01 | 10.81 | G1 | 9.19 | 1200.00 | 130.00 | 150.00 |
H1 | 108.95 | 11.01 | H1 | 8.99 | 1200.00 | 130.00 | 150.00 |
A2 | 101.77 | 11.79 | A2 | 8.21 | 1200.00 | 130.00 | 150.00 |
B2 | 109.05 | 11.00 | B2 | 9.00 | 1200.00 | 130.00 | 150.00 |
C2 | 106.42 | 11.28 | C2 | 8.72 | 1200.00 | 130.00 | 150.00 |
D2 | 94.38 | 12.71 | D2 | 7.29 | 1200.00 | 130.00 | 150.00 |
E2 | 84.67 | 14.17 | E2 | 5.83 | 1200.00 | 130.00 | 150.00 |
F2 | 108.48 | 11.06 | F2 | 8.94 | 1200.00 | 130.00 | 150.00 |
G2 | 62.01 | 19.35 | G2 | 0.65 | 1200.00 | 130.00 | 150.00 |
H2* | 57.54 | 20.00 | H2* | 0.00 | 1150.80 | 130.00 | 150.00 |
A3 | 104.16 | 11.52 | A3 | 8.48 | 1200.00 | 130.00 | 150.00 |
B3 | 128.33 | 9.35 | B3 | 10.65 | 1200.00 | 130.00 | 150.00 |
C3 | 90.52 | 13.26 | C3 | 6.74 | 1200.00 | 130.00 | 150.00 |
D3 | 75.38 | 15.92 | D3 | 4.08 | 1200.00 | 130.00 | 150.00 |
E3 | 139.81 | 8.58 | E3 | 11.42 | 1200.00 | 130.00 | 150.00 |
F3 | 67.75 | 17.71 | F3 | 2.29 | 1200.00 | 130.00 | 150.00 |
G3 | 98.83 | 12.14 | G3 | 7.86 | 1200.00 | 130.00 | 150.00 |
H3 | 83.68 | 14.34 | H3 | 5.66 | 1200.00 | 130.00 | 150.00 |
A4 | 89.42 | 13.42 | A4 | 6.58 | 1200.00 | 130.00 | 150.00 |
B4 | 80.22 | 14.96 | B4 | 5.04 | 1200.00 | 130.00 | 150.00 |
C4 | 114.26 | 10.50 | C4 | 9.50 | 1200.00 | 130.00 | 150.00 |
D4 | 119.02 | 10.08 | D4 | 9.92 | 1200.00 | 130.00 | 150.00 |
E4 | 90.16 | 13.31 | E4 | 6.69 | 1200.00 | 130.00 | 150.00 |
F4 | 117.73 | 10.19 | F4 | 9.81 | 1200.00 | 130.00 | 150.00 |
G4 | 133.36 | 9.00 | G4 | 11.00 | 1200.00 | 130.00 | 150.00 |
H4 | 90.89 | 13.20 | H4 | 6.80 | 1200.00 | 130.00 | 150.00 |
A5 | 108.53 | 11.06 | A5 | 8.94 | 1200.00 | 130.00 | 150.00 |
B5 | 108.85 | 11.02 | B5 | 8.98 | 1200.00 | 130.00 | 150.00 |
C5 | 100.28 | 11.97 | C5 | 8.03 | 1200.00 | 130.00 | 150.00 |
D5 | 109.51 | 10.96 | D5 | 9.04 | 1200.00 | 130.00 | 150.00 |
E5 | 98.92 | 12.13 | E5 | 7.87 | 1200.00 | 130.00 | 150.00 |
F5 | 114.30 | 10.50 | F5 | 9.50 | 1200.00 | 130.00 | 150.00 |
G5 | 166.66 | 7.20 | G5 | 12.80 | 1200.00 | 130.00 | 150.00 |
H5 | 100.31 | 11.96 | H5 | 8.04 | 1200.00 | 130.00 | 150.00 |
A6 | 98.50 | 12.18 | A6 | 7.82 | 1200.00 | 130.00 | 150.00 |
B6 | 74.95 | 16.01 | B6 | 3.99 | 1200.00 | 130.00 | 150.00 |
C6 | 91.90 | 13.06 | C6 | 6.94 | 1200.00 | 130.00 | 150.00 |
D6 | 63.31 | 18.95 | D6 | 1.05 | 1200.00 | 130.00 | 150.00 |
E6 | 76.35 | 15.72 | E6 | 4.28 | 1200.00 | 130.00 | 150.00 |
F6 | 77.34 | 15.52 | F6 | 4.48 | 1200.00 | 130.00 | 150.00 |
G6 | 100.09 | 11.99 | G6 | 8.01 | 1200.00 | 130.00 | 150.00 |
H6 | 76.76 | 15.63 | H6 | 4.37 | 1200.00 | 130.00 | 150.00 |
A7 | 71.86 | 16.70 | A7 | 3.30 | 1200.00 | 130.00 | 150.00 |
B7** | 39.82 | 20.00 | B7** | 0.00 | 796.40 | 130.00 | 150.00 |
C7 | 79.72 | 15.05 | C7 | 4.95 | 1200.00 | 130.00 | 150.00 |
D7 | 100.77 | 11.91 | D7 | 8.09 | 1200.00 | 130.00 | 150.00 |
E7 | 197.06 | 6.09 | E7 | 13.91 | 1200.00 | 130.00 | 150.00 |
F7 | 60.37 | 19.88 | F7 | 0.12 | 1200.00 | 130.00 | 150.00 |
G7* | 53.66 | 20.00 | G7* | 0.00 | 1073.20 | 130.00 | 150.00 |
H7* | 55.88 | 20.00 | H7* | 0.00 | 1117.60 | 130.00 | 150.00 |
A8* | 59.88 | 20.00 | A8* | 0.00 | 1197.60 | 130.00 | 150.00 |
B8 | 85.67 | 14.01 | B8 | 5.99 | 1200.00 | 130.00 | 150.00 |
C8 | 96.65 | 12.42 | C8 | 7.58 | 1200.00 | 130.00 | 150.00 |
D8 | 128.37 | 9.35 | D8 | 10.65 | 1200.00 | 130.00 | 150.00 |
E8 | 89.01 | 13.48 | E8 | 6.52 | 1200.00 | 130.00 | 150.00 |
F8 | 95.33 | 12.59 | F8 | 7.41 | 1200.00 | 130.00 | 150.00 |
G8 | 84.04 | 14.28 | G8 | 5.72 | 1200.00 | 130.00 | 150.00 |
H8 | 144.05 | 8.33 | H8 | 11.67 | 1200.00 | 130.00 | 150.00 |
A9 | 76.42 | 15.70 | A9 | 4.30 | 1200.00 | 130.00 | 150.00 |
B9 | 228.10 | 5.26 | B9 | 14.74 | 1200.00 | 130.00 | 150.00 |
C9 | 119.19 | 10.07 | C9 | 9.93 | 1200.00 | 130.00 | 150.00 |
D9 | 269.45 | 4.45 | D9 | 15.55 | 1200.00 | 130.00 | 150.00 |
E9* | 49.91 | 20.00 | E9* | 0.00 | 998.20 | 130.00 | 150.00 |
F9 | 86.00 | 13.95 | F9 | 6.05 | 1200.00 | 130.00 | 150.00 |
G9*** | 593.63 | 2.02 | G9*** | 17.98 | 1200.00 | 130.00 | 150.00 |
H9* | 51.54 | 20.00 | H9* | 0.00 | 1030.80 | 130.00 | 150.00 |
A10 | 59.93 | 20.00 | A10 | 0.00 | 1198.60 | 130.00 | 150.00 |
B10 | 72.25 | 16.61 | B10 | 3.39 | 1200.00 | 130.00 | 150.00 |
C10 | 69.85 | 17.18 | C10 | 2.82 | 1200.00 | 130.00 | 150.00 |
D10 | 129.33 | 9.28 | D10 | 10.72 | 1200.00 | 130.00 | 150.00 |
E10 | 62.05 | 19.34 | E10 | 0.66 | 1200.00 | 130.00 | 150.00 |
F10 | 139.09 | 8.63 | F10 | 11.37 | 1200.00 | 130.00 | 150.00 |
G10 | 189.64 | 6.33 | G10 | 13.67 | 1200.00 | 130.00 | 150.00 |
H10 | 70.48 | 17.03 | H10 | 2.97 | 1200.00 | 130.00 | 150.00 |
A11 | 152.47 | 7.87 | A11 | 12.13 | 1200.00 | 130.00 | 150.00 |
B11 | 90.51 | 13.26 | B11 | 6.74 | 1200.00 | 130.00 | 150.00 |
C11 | 106.38 | 11.28 | C11 | 8.72 | 1200.00 | 130.00 | 150.00 |
D11 | 88.70 | 13.53 | D11 | 6.47 | 1200.00 | 130.00 | 150.00 |
E11 | 78.12 | 15.36 | E11 | 4.64 | 1200.00 | 130.00 | 150.00 |
F11 | 61.46 | 19.52 | F11 | 0.48 | 1200.00 | 130.00 | 150.00 |
G11 | 111.77 | 10.74 | G11 | 9.26 | 1200.00 | 130.00 | 150.00 |
H11 | 125.43 | 9.57 | H11 | 10.43 | 1200.00 | 130.00 | 150.00 |
A12 | 74.20 | 16.17 | A12 | 3.83 | 1200.00 | 130.00 | 150.00 |
B12 | 92.20 | 13.02 | B12 | 6.98 | 1200.00 | 130.00 | 150.00 |
C12** | 46.85 | 20.00 | C12* | 0.00 | 937.00 | 130.00 | 150.00 |
D12 | 68.39 | 17.55 | D12 | 2.45 | 1200.00 | 130.00 | 150.00 |
E12 | 83.77 | 14.32 | E12 | 5.68 | 1200.00 | 130.00 | 150.00 |
F12* | 53.64 | 20.00 | F12* | 0.00 | 1072.80 | 130.00 | 150.00 |
G12 | 106.40 | 11.28 | G12 | 8.72 | 1200.00 | 130.00 | 150.00 |
H12 | 74.57 | 16.09 | H12 | 3.91 | 1200.00 | 130.00 | 150.00 |
- Eluted volume 22ul with Elution buffer. After Qubit ss-DNA assay the total yields will be calculated based on ~17ul left in the well.
- Note:
- *= Total amount in bis-cvt lower than 1200ng, but still above 1000ng
- ** or ***= Performed two bis-cvt (one in the plate and one in the column (B7 (conc, too low, G9 (conc. too high)
- ***= conc. too high
Qubit ssDNA assay
- Follow protocol provided with the kit
- Took 1ul of bis-cvt DNA into 9ul of H2O, then added the diluted DNA to 190ul ssDNA Qubit reagent (dilution factor = 200)
Well ID | Concentration in the Qubit (ng/mL) | Volume (ul) | Final conc. (ng/ul) | Yields (ng) |
A1 | 145 | 17 | 29 | 493 |
B1 | 116 | 17 | 23.2 | 394.4 |
C1 | 158 | 17 | 31.6 | 537.2 |
D1 | 165 | 17 | 33 | 561 |
E1 | 104 | 17 | 20.8 | 353.6 |
F1 | 94.9 | 17 | 18.98 | 322.66 |
G1 | 144 | 17 | 28.8 | 489.6 |
H1 | 118 | 17 | 23.6 | 401.2 |
A2 | 96.7 | 17 | 19.34 | 328.78 |
B2 | 128 | 17 | 25.6 | 435.2 |
C2 | 125 | 17 | 25 | 425 |
D2 | 116 | 17 | 23.2 | 394.4 |
E2 | 119 | 17 | 23.8 | 404.6 |
F2 | 160 | 17 | 32 | 544 |
G2 | 94.6 | 17 | 18.92 | 321.64 |
H2 | 103 | 17 | 20.6 | 350.2 |
A3 | 87.8 | 17 | 17.56 | 298.52 |
B3 | 145 | 17 | 29 | 493 |
C3 | 145 | 17 | 29 | 493 |
D3 | 108 | 17 | 21.6 | 367.2 |
E3 | 129 | 17 | 25.8 | 438.6 |
F3 | 116 | 17 | 23.2 | 394.4 |
G3 | 153 | 17 | 30.6 | 520.2 |
H3 | 140 | 17 | 28 | 476 |
A4 | 113 | 17 | 22.6 | 384.2 |
B4 | 132 | 17 | 26.4 | 448.8 |
C4 | 120 | 17 | 24 | 408 |
D4 | 142 | 17 | 28.4 | 482.8 |
E4 | 128 | 17 | 25.6 | 435.2 |
F4 | 132 | 17 | 26.4 | 448.8 |
G4 | 117 | 17 | 23.4 | 397.8 |
H4 | 119 | 17 | 23.8 | 404.6 |
A5 | 126 | 17 | 25.2 | 428.4 |
B5 | 122 | 17 | 24.4 | 414.8 |
C5 | 111 | 17 | 22.2 | 377.4 |
D5 | 119 | 17 | 23.8 | 404.6 |
E5 | 128 | 17 | 25.6 | 435.2 |
F5 | 172 | 17 | 34.4 | 584.8 |
G5 | 112 | 17 | 22.4 | 380.8 |
H5 | 72 | 17 | 14.4 | 244.8 |
A6 | 133 | 17 | 26.6 | 452.2 |
B6 | 104 | 17 | 20.8 | 353.6 |
C6 | 118 | 17 | 23.6 | 401.2 |
D6 | 101 | 17 | 20.2 | 343.4 |
E6 | 96.3 | 17 | 19.26 | 327.42 |
F6 | 104 | 17 | 20.8 | 353.6 |
G6 | 135 | 17 | 27 | 459 |
H6 | 111 | 17 | 22.2 | 377.4 |
A7 | 169 | 17 | 33.8 | 574.6 |
B7 | 115 | 25 | 23 | 575 |
C7 | 148 | 17 | 29.6 | 503.2 |
D7 | 171 | 17 | 34.2 | 581.4 |
E7 | 168 | 17 | 33.6 | 571.2 |
F7 | 137 | 17 | 27.4 | 465.8 |
G7 | 139 | 17 | 27.8 | 472.6 |
H7 | 123 | 17 | 24.6 | 418.2 |
A8 | 142 | 17 | 28.4 | 482.8 |
B8 | 123 | 17 | 24.6 | 418.2 |
C8 | 158 | 17 | 31.6 | 537.2 |
D8 | 79.8 | 17 | 15.96 | 271.32 |
E8 | 181 | 17 | 36.2 | 615.4 |
F8 | 90 | 17 | 18 | 306 |
G8 | 167 | 17 | 33.4 | 567.8 |
H8 | 190 | 17 | 38 | 646 |
A9 | 167 | 17 | 33.4 | 567.8 |
B9 | 202 | 17 | 40.4 | 686.8 |
C9 | 178 | 17 | 35.6 | 605.2 |
D9 | 249 | 17 | 49.8 | 846.6 |
E9 | 115 | 17 | 23 | 391 |
F9 | 155 | 17 | 31 | 527 |
G9 | 264 | 25 | 52.8 | 1320 |
H9 | 125 | 17 | 25 | 425 |
A10 | 148 | 17 | 29.6 | 503.2 |
B10 | 166 | 17 | 33.2 | 564.4 |
C10 | 178 | 17 | 35.6 | 605.2 |
D10 | 187 | 17 | 37.4 | 635.8 |
E10 | 155 | 17 | 31 | 527 |
F10 | 87 | 17 | 17.4 | 295.8 |
G10 | 194 | 17 | 38.8 | 659.6 |
H10 | 165 | 17 | 33 | 561 |
A11 | 203 | 17 | 40.6 | 690.2 |
B11 | 168 | 17 | 33.6 | 571.2 |
C11 | 71.6 | 17 | 14.32 | 243.44 |
D11 | 165 | 17 | 33 | 561 |
E11 | 147 | 17 | 29.4 | 499.8 |
F11 | 123 | 17 | 24.6 | 418.2 |
G11 | 111 | 17 | 22.2 | 377.4 |
H11 | 184 | 17 | 36.8 | 625.6 |
A12 | 136 | 17 | 27.2 | 462.4 |
B12 | 169 | 17 | 33.8 | 574.6 |
C12 | 135 | 25 | 27 | 675 |
D12 | 177 | 17 | 35.4 | 601.8 |
E12 | 177 | 17 | 35.4 | 601.8 |
F12 | 132 | 17 | 26.4 | 448.8 |
G12 | 130 | 17 | 26 | 442 |
H12 | 230 | 17 | 46 | 782 |
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 | 0.39 | 0.29 | 30.15 | DMR.s1 |
DMR.s2 | 0.07 | 2.24 | 0.16 | 0.35 | 0.47 | 48.85 | DMR.s2 |
DMR.s3 | 0.23 | 18.90 | 4.41 | 23.20 | 0.19 | 19.94 | DMR.s3 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 62.90 | 0.30 | 31.55 | DMR.s4 |
DMR.s5 | 4.83 | 18.90 | 91.29 | 52.70 | 1.73 | 181.88 | DMR.s5 |
DMR.Exp1 | 0.23 | 18.90 | 4.44 | 31.88 | 0.14 | 14.61 | DMR.Exp1 |
DMR.Exp2 | 0.19 | 18.90 | 3.50 | 24.11 | 0.15 | 15.26 | DMR.Exp2 |
DMR.Exp3 | 0.27 | 18.90 | 5.03 | 28.90 | 0.17 | 18.26 | DMR.Exp3 |
1.05uM Oligo suppressor-set1 | 0.15 | 15.75 | 1.05uM Oligo suppressor-set1 | ||||
1.05uM Oligo suppressor-set2 | 0.15 | 15.75 | 1.05uM Oligo suppressor-set2 | ||||
Total volume | only probe | 3.43 |
Reaction mix
Components | 1rxn (ul) | 105 rxn mix (ul) |
10X AmpLigase Buffer | 2.50 | 262.50 |
DMR330k probe mix (ul) | 3.43 | 360.15 |
105nM Oligo suppressor | 0.30 | 31.50 |
Total volume (ul) | 6.23 | 654.15 |
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 | 29.00 | 10.34 | A1 | 8.43 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B1 | 23.20 | 12.93 | B1 | 5.84 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C1 | 31.60 | 9.49 | C1 | 9.28 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D1 | 33.00 | 9.09 | D1 | 9.68 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E1 | 20.80 | 14.42 | E1 | 4.35 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F1 | 18.98 | 15.81 | F1 | 2.96 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G1 | 28.80 | 10.42 | G1 | 8.35 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H1 | 23.60 | 12.71 | H1 | 6.06 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A2 | 19.34 | 15.51 | A2 | 3.26 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B2 | 25.60 | 11.72 | B2 | 7.05 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C2 | 25.00 | 12.00 | C2 | 6.77 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D2 | 23.20 | 12.93 | D2 | 5.84 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E2 | 23.80 | 12.61 | E2 | 6.16 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F2 | 32.00 | 9.38 | F2 | 9.40 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G2 | 18.92 | 15.86 | G2 | 2.91 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H2 | 20.60 | 14.56 | H2 | 4.21 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A3 | 17.56 | 17.08 | A3 | 1.69 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B3 | 29.00 | 10.34 | B3 | 8.43 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C3 | 29.00 | 10.34 | C3 | 8.43 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D3 | 21.60 | 13.89 | D3 | 4.88 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E3 | 25.80 | 11.63 | E3 | 7.14 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F3 | 23.20 | 12.93 | F3 | 5.84 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G3 | 30.60 | 9.80 | G3 | 8.97 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H3 | 28.00 | 10.71 | H3 | 8.06 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A4 | 22.60 | 13.27 | A4 | 5.50 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B4 | 26.40 | 11.36 | B4 | 7.41 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C4 | 24.00 | 12.50 | C4 | 6.27 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D4 | 28.40 | 10.56 | D4 | 8.21 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E4 | 25.60 | 11.72 | E4 | 7.05 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F4 | 26.40 | 11.36 | F4 | 7.41 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G4 | 23.40 | 12.82 | G4 | 5.95 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H4 | 23.80 | 12.61 | H4 | 6.16 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A5 | 25.20 | 11.90 | A5 | 6.87 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B5 | 24.40 | 12.30 | B5 | 6.47 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C5 | 22.20 | 13.51 | C5 | 5.26 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D5 | 23.80 | 12.61 | D5 | 6.16 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E5 | 25.60 | 11.72 | E5 | 7.05 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F5 | 34.40 | 8.72 | F5 | 10.05 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G5 | 22.40 | 13.39 | G5 | 5.38 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H5 | 14.40 | 17.00 | H5** | 1.77 | 244.80 | 3.43 | 2.50 | 0.30 | 25.00 |
A6 | 26.60 | 11.28 | A6 | 7.49 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B6 | 20.80 | 14.42 | B6 | 4.35 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C6 | 23.60 | 12.71 | C6 | 6.06 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D6 | 20.20 | 14.85 | D6 | 3.92 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E6 | 19.26 | 15.58 | E6 | 3.19 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F6 | 20.80 | 14.42 | F6 | 4.35 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G6 | 27.00 | 11.11 | G6 | 7.66 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H6 | 22.20 | 13.51 | H6 | 5.26 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A7 | 33.80 | 8.88 | A7 | 9.89 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B7 | 23.00 | 13.04 | B7 | 5.73 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C7 | 29.60 | 10.14 | C7 | 8.63 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D7 | 34.20 | 8.77 | D7 | 10.00 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E7 | 33.60 | 8.93 | E7 | 9.84 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F7 | 27.40 | 10.95 | F7 | 7.82 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G7 | 27.80 | 10.79 | G7 | 7.98 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H7 | 24.60 | 12.20 | H7 | 6.57 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A8 | 28.40 | 10.56 | A8 | 8.21 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B8 | 24.60 | 12.20 | B8 | 6.57 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C8 | 31.60 | 9.49 | C8 | 9.28 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D8 | 15.96 | 17.00 | D8** | 1.77 | 271.32 | 3.43 | 2.50 | 0.30 | 25.00 |
E8 | 36.20 | 8.29 | E8 | 10.48 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F8 | 18.00 | 16.67 | F8 | 2.10 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G8 | 33.40 | 8.98 | G8 | 9.79 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H8 | 38.00 | 7.89 | H8 | 10.88 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A9 | 33.40 | 8.98 | A9 | 9.79 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B9 | 40.40 | 7.43 | B9 | 11.34 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C9 | 35.60 | 8.43 | C9 | 10.34 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D9 | 49.80 | 6.02 | D9 | 12.75 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E9 | 23.00 | 13.04 | E9 | 5.73 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F9 | 31.00 | 9.68 | F9 | 9.09 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G9 | 52.80 | 5.68 | G9 | 13.09 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H9 | 25.00 | 12.00 | H9 | 6.77 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A10 | 29.60 | 10.14 | A10 | 8.63 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B10 | 33.20 | 9.04 | B10 | 9.73 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C10 | 35.60 | 8.43 | C10 | 10.34 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D10 | 37.40 | 8.02 | D10 | 10.75 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E10 | 31.00 | 9.68 | E10 | 9.09 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F10 | 17.40 | 17.00 | F10** | 1.77 | 295.80 | 3.43 | 2.50 | 0.30 | 25.00 |
G10 | 38.80 | 7.73 | G10 | 11.04 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H10 | 33.00 | 9.09 | H10 | 9.68 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A11 | 40.60 | 7.39 | A11 | 11.38 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B11 | 33.60 | 8.93 | B11 | 9.84 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C11 | 14.32 | 17.00 | C11** | 1.77 | 243.44 | 3.43 | 2.50 | 0.30 | 25.00 |
D11 | 33.00 | 9.09 | D11 | 9.68 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E11 | 29.40 | 10.20 | E11 | 8.57 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F11 | 24.60 | 12.20 | F11 | 6.57 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G11 | 22.20 | 13.51 | G11 | 5.26 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H11 | 36.80 | 8.15 | H11 | 10.62 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
A12 | 27.20 | 11.03 | A12 | 7.74 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
B12 | 33.80 | 8.88 | B12 | 9.89 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
C12 | 27.00 | 11.11 | C12 | 7.66 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
D12 | 35.40 | 8.47 | D12 | 10.30 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
E12 | 35.40 | 8.47 | E12 | 10.30 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
F12 | 26.40 | 11.36 | F12 | 7.41 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
G12 | 26.00 | 11.54 | G12 | 7.23 | 300.00 | 3.43 | 2.50 | 0.30 | 25.00 |
H12 | 46.00 | 6.52 | H12 | 12.25 | 300.00 | 3.43 | 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): 81.77ul
- Aliquot reaction mix 6.23ul with multichannel pipette and mix well
- Put mineral oil in basin ~3 mL)
- Layer the capture reaction with 25ul 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_17) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_18)
SLN mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare 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 ul to each well with multichannel pipettes
ExoI/III preparation
- Mix 110 ul each of ExoI and ExoIII and aliquot 27.5ul into striptube
- Add 2ul to each well with multichannel pipettes
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX No.1-96)
- Spin the capture plate for 3 min to collect all liquid to the bottom of the well
- To make the sequencing library, use at least 2/3 of the total volume for amplification (total volume =25ul +2.5+2 = 29.5ul).
- I alway use 8-10 ul in each PCR reaction total volume 100ul by trying out the first PCR with few samples in total volume 50ul to see the qPCR curve and the number of cycle then do the whole plate.
- Sample to be tested, A1-D1
Components | 1rxn | 4.2 rxn mix |
Captured template | 4.00 | 0.00 |
10uM AmpF6.4Sol | 1.00 | 4.20 |
10uM AmpR6.3 (IndX, X=1-4) | 1.00 | 0 |
50x SYBG | 0.40 | 1.68 |
2x Phusion MM | 25.00 | 105.00 |
H2O | 18.60 | 78.12 |
Total | 50.00 | Aliquot 45ul, add 10ul template and 1 ul of AmpR6.3 IndX |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
Components | 1rxn | 100 rxn mix |
Captured template | 10.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 200.00 |
10uM AmpR6.3 (IndX, X=1-96) | 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)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
Plate layout and barcode primer
==A1== | ===A2== | ===A3=== | ===A4=== | ===A5=== | ===A6=== | ===A===7 | ===A8=== | ===A9=== | ===A10=== | ===A11=== | ===A12=== |
Ind1 | Ind9 | Ind17 | Ind25 | Ind33 | Ind41 | Ind49 | Ind57 | Ind65 | Ind73 | Ind81 | Ind89 |
B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | B11 | B12 |
Ind2 | Ind10 | Ind18 | Ind26 | Ind34 | Ind42 | Ind50 | Ind58 | Ind66 | Ind74 | Ind82 | Ind90 |
C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | C11 | C12 |
Ind3 | Ind11 | Ind19 | Ind27 | Ind35 | Ind43 | Ind51 | Ind59 | Ind67 | Ind75 | Ind83 | Ind91 |
D1 | D2 | D3 | D4 | D5 | D6 | D7 | D8 | D9 | D10 | D11 | D12 |
Ind4 | Ind12 | Ind20 | Ind28 | Ind36 | Ind44 | Ind52 | Ind60 | Ind68 | Ind76 | Ind84 | Ind92 |
E1 | E2 | E3 | E4 | E5 | E6 | E7 | E8 | E9 | E10 | E11 | E12 |
Ind5 | Ind13 | Ind21 | Ind29 | Ind37 | Ind45 | Ind53 | Ind61 | Ind69 | Ind77 | Ind85 | Ind93 |
F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 | F10 | F11 | F12 |
Ind6 | Ind14 | Ind22 | Ind30 | Ind38 | Ind46 | Ind54 | Ind62 | Ind70 | Ind78 | Ind86 | Ind94 |
G1 | G2 | G3 | G4 | G5 | G6 | G7 | G8 | G9 | G10 | G11 | G12 |
Ind7 | Ind15 | Ind23 | Ind31 | Ind39 | Ind47 | Ind55 | Ind63 | Ind71 | Ind79 | Ind87 | Ind95 |
H1 | H2 | H3 | H4 | H5 | H6 | H7 | H8 | H9 | H10 | H11 | H12 |
Ind8 | Ind16 | Ind24 | Ind32 | Ind40 | Ind48 | Ind56 | Ind64 | Ind72 | Ind80 | Ind88 | Ind96 |