Noi/NOTES/2012-5-21
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DMR 330k BSPP capture of samples from Sergio[edit]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-20#Bisulfite_conversion
Repeat bisulfite conversion[edit]
Reagent prep Add the following reagents to CT Conversion Reagent - H2O 9.0mL - M-Dissolving Buffer 500ul - M-Dilution Buffer 3.0mL Mix at room temperature with frequent vortexing or shaking for 15 minutes Add 144 ml of 100% ethanol to the 36 ml M-Wash Buffer concentrate before use
Lab IDs | Conc. (ng/ul) | Volume for 1200ng | LabID ID | Well IDs | H2O | Total amount (ng) | CT conversion reagent (ul) | Total volume (ul) |
Ap15-1 | 110.0 | 10.91 | Ap15-1 | A1 | 9.09 | 1200.00 | 130.00 | 150.00 |
Ap15-2 | 139.1 | 8.63 | Ap15-2 | B1 | 11.37 | 1200.00 | 130.00 | 150.00 |
Ap15-3 | 145.1 | 8.27 | Ap15-3 | C1 | 11.73 | 1200.00 | 130.00 | 150.00 |
Ap15-4 | 295.5 | 4.06 | Ap15-4 | D1 | 15.94 | 1200.00 | 130.00 | 150.00 |
Ap15-5 | 128.1 | 9.37 | Ap15-5 | E1 | 10.63 | 1200.00 | 130.00 | 150.00 |
Ap15-6 | 153.9 | 7.80 | Ap15-6 | F1 | 12.20 | 1200.00 | 130.00 | 150.00 |
Ap15-7 | 183.5 | 6.54 | Ap15-7 | G1 | 13.46 | 1200.00 | 130.00 | 150.00 |
Ap15-8 | 149.3 | 8.04 | Ap15-8 | H1 | 11.96 | 1200.00 | 130.00 | 150.00 |
Ap15-9 | 111.2 | 10.79 | Ap15-9 | A2 | 9.21 | 1200.00 | 130.00 | 150.00 |
Ap15-10 | 153.5 | 7.82 | Ap15-10 | B2 | 12.18 | 1200.00 | 130.00 | 150.00 |
Ap15-11 | 87.5 | 13.71 | Ap15-11 | C2 | 6.29 | 1200.00 | 130.00 | 150.00 |
Ap15-12 | 109.7 | 10.94 | Ap15-12 | D2 | 9.06 | 1200.00 | 130.00 | 150.00 |
Ap15-13 | 191.3 | 6.27 | Ap15-13 | E2 | 13.73 | 1200.00 | 130.00 | 150.00 |
Ap15-14 | 108.7 | 11.04 | Ap15-14 | F2 | 8.96 | 1200.00 | 130.00 | 150.00 |
Ap15-15 | 134.2 | 8.94 | Ap15-15 | G2 | 11.06 | 1200.00 | 130.00 | 150.00 |
Ap15-16 | 50.2 | 20.00 | Ap15-16 | H2 | 0.00 | 1200.00 | 130.00 | 150.00 |
Ap15-17 | 183.0 | 6.56 | Ap15-17 | A3 | 13.44 | 1200.00 | 130.00 | 150.00 |
Ap15-18 | 82.6 | 14.53 | Ap15-18 | B3 | 5.47 | 1200.00 | 130.00 | 150.00 |
Ap15-19 | 263.4 | 4.56 | Ap15-19 | C3 | 15.44 | 1200.00 | 130.00 | 150.00 |
Ma15-1 | 86.7 | 13.84 | Ma15-1 | D3 | 6.16 | 1200.00 | 130.00 | 150.00 |
Ma15-2 | 215.2 | 5.58 | Ma15-2 | E3 | 14.42 | 1200.00 | 130.00 | 150.00 |
Ma15-3 | 252.0 | 4.76 | Ma15-3 | F3 | 15.24 | 1200.00 | 130.00 | 150.00 |
Ma15-4 | 226.1 | 5.31 | Ma15-4 | G3 | 14.69 | 1200.00 | 130.00 | 150.00 |
Ma15-5 | 238.7 | 5.03 | Ma15-5 | H3 | 14.97 | 1200.00 | 130.00 | 150.00 |
Ma15-6 | 83.4 | 14.39 | Ma15-6 | A4 | 5.61 | 1200.00 | 130.00 | 150.00 |
Ma15-7 | 236.8 | 5.07 | Ma15-7 | B4 | 14.93 | 1200.00 | 130.00 | 150.00 |
Ma15-8 | 175.9 | 6.82 | Ma15-8 | C4 | 13.18 | 1200.00 | 130.00 | 150.00 |
Ma15-9 | 164.3 | 7.30 | Ma15-9 | D4 | 12.70 | 1200.00 | 130.00 | 150.00 |
Ma15-10 | 205.9 | 5.83 | Ma15-10 | E4 | 14.17 | 1200.00 | 130.00 | 150.00 |
Ma15-11 | 97.6 | 12.30 | Ma15-11 | F4 | 7.70 | 1200.00 | 130.00 | 150.00 |
Ma15-12 | 129.5 | 9.27 | Ma15-12 | G4 | 10.73 | 1200.00 | 130.00 | 150.00 |
Ma15-13 | 263.1 | 4.56 | Ma15-13 | H4 | 15.44 | 1200.00 | 130.00 | 150.00 |
Ma15-14 | 76.1 | 15.77 | Ma15-14 | A5 | 4.23 | 1200.00 | 130.00 | 150.00 |
Ma15-15 | 138.7 | 8.65 | Ma15-15 | B5 | 11.35 | 1200.00 | 130.00 | 150.00 |
Ma15-16 | 216.4 | 5.55 | Ma15-16 | C5 | 14.45 | 1200.00 | 130.00 | 150.00 |
Ma15-17 | 137.6 | 8.72 | Ma15-17 | D5 | 11.28 | 1200.00 | 130.00 | 150.00 |
Ma15-18 | 224.6 | 5.34 | Ma15-18 | E5 | 14.66 | 1200.00 | 130.00 | 150.00 |
Ma15-19 | 111.7 | 10.74 | Ma15-19 | F5 | 9.26 | 1200.00 | 130.00 | 150.00 |
Ma15-20 | 289.9 | 4.14 | Ma15-20 | G5 | 15.86 | 1200.00 | 130.00 | 150.00 |
Ma15-21 | 172.9 | 6.94 | Ma15-21 | H5 | 13.06 | 1200.00 | 130.00 | 150.00 |
Lab IDs | Conc. (ng/ul) | Volume for 1200ng | LabID ID | Well IDs | H2O | Total amount (ng) | CT conversion reagent (ul) | Total volume (ul) |
SR-2 | 90.8 | 13.22 | SR-2 | B6 | 6.78 | 1200.00 | 130.00 | 150.00 |
SR-10 | 184.8 | 6.49 | SR-10 | B7 | 13.51 | 1200.00 | 130.00 | 150.00 |
SR-11 | 105.4 | 5.00 | SR-11 | C7 | 15.00 | 527.00 | 130.00 | 150.00 |
SR-12 | 181.4 | 6.62 | SR-12 | D7 | 13.38 | 1200.00 | 130.00 | 150.00 |
SR-13 | 270.0 | 4.44 | SR-13 | E7 | 15.56 | 1200.00 | 130.00 | 150.00 |
SR-14 | 33.0 | 20.00 | SR-14 | F7 | 0.00 | 495.00 | 130.00 | 150.00 |
SR-15 | 67.0 | 17.91 | SR-15 | G7 | 2.09 | 1200.00 | 130.00 | 150.00 |
SR-16 | 145.6 | 8.24 | SR-16 | H7 | 11.76 | 1200.00 | 130.00 | 150.00 |
SR-17 | 245.3 | 4.89 | SR-17 | A8 | 15.11 | 1200.00 | 130.00 | 150.00 |
SR-19 | 159.5 | 7.52 | SR-19 | C8 | 12.48 | 1200.00 | 130.00 | 150.00 |
SR-20 | 195.7 | 6.13 | SR-20 | D8 | 13.87 | 1200.00 | 130.00 | 150.00 |
Well IDs/Sample IDs | Well IDs/Sample IDs | Well IDs/Sample IDs | Well IDs/Sample IDs | Well IDs/Sample IDs | Well IDs/Sample IDs | Well IDs/Sample IDs | Well IDs/Sample IDs |
A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 |
Ap15-1 | Ap15-9 | Ap15-17 | Ma15-6 | Ma15-14 | SR-1 => NO | SR-9 | SR-17 |
B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 |
Ap15-2 | Ap15-10 | Ap15-18 | Ma15-7 | Ma15-15 | SR-2 | SR-10 | SR-18 => NO |
C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 |
Ap15-3 | Ap15-11 | Ap15-19 | Ma15-8 | Ma15-16 | SR-3 => NO | SR-11 | SR-19 |
D1 | D2 | D3 | D4 | D5 | D6 | D7 | D8 |
Ap15-4 | Ap15-12 | Ma15-1 | Ma15-9 | Ma15-17 | SR-4 => NO | SR-12 | SR-20 |
E1 | E2 | E3 | E4 | E5 | E6 | E7 | E8 |
Ap15-5 | Ap15-13 | Ma15-2 | Ma15-10 | Ma15-18 | SR-5 => NO | SR-13 | SR-21 => NO |
F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 |
Ap15-6 | Ap15-14 | Ma15-3 | Ma15-11 | Ma15-19 | SR-6 => NO | SR-14 | SR-22 => NO |
G1 | G2 | G3 | G4 | G5 | G6 | G7 | G8 |
Ap15-7 | Ap15-15 | Ma15-4 | Ma15-12 | Ma15-20 | SR-7 => NO | SR-15 | |
H1 | H2 | H3 | H4 | H5 | H6 | H7 | H8 |
Ap15-8 | Ap15-16 | Ma15-5 | Ma15-13 | Ma15-21 | SR-8 => NO | SR-16 |
Note: no more gDNA for SR-14 samples Program 98°C for 10 minutes 64°C for 2.5 hours 4°C storage up to 20 hours Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA)
Qubit ssDNA assay[edit]
- I combined bis-cvt gDNA of two batch together and measure concentration.
- Sample in assay 1ul, dilution 200x
Sample | Concentration in the Qubit | ' | Final conc. (ng/ul) | Yields in 30ul (ng) |
A1 | 50.6 | ng/mL | 10.12 | 303.60 |
B1 | 47.9 | ng/mL | 9.58 | 287.40 |
C1 | 56.9 | ng/mL | 11.38 | 341.40 |
D1 | 63.2 | ng/mL | 12.64 | 379.20 |
E1 | 47.1 | ng/mL | 9.42 | 282.60 |
F1 | 66.2 | ng/mL | 13.24 | 397.20 |
G1 | 67.1 | ng/mL | 13.42 | 402.60 |
H1 | 59.7 | ng/mL | 11.94 | 358.20 |
A2 | 69 | ng/mL | 13.80 | 414.00 |
B2 | 53.2 | ng/mL | 10.64 | 319.20 |
C2 | 52.8 | ng/mL | 10.56 | 316.80 |
D2 | 55.7 | ng/mL | 11.14 | 334.20 |
E2 | 58.5 | ng/mL | 11.70 | 351.00 |
F2 | 60.9 | ng/mL | 12.18 | 365.40 |
G2 | 51.2 | ng/mL | 10.24 | 307.20 |
H2 | 36.8 | ng/mL | 7.36 | 220.80 |
A3 | 71.5 | ng/mL | 14.30 | 429.00 |
B3 | 57.5 | ng/mL | 11.50 | 345.00 |
C3 | 79.4 | ng/mL | 15.88 | 476.40 |
D3 | 44.2 | ng/mL | 8.84 | 265.20 |
E3 | 43 | ng/mL | 8.60 | 258.00 |
F3 | 58.1 | ng/mL | 11.62 | 348.60 |
G3 | 49.4 | ng/mL | 9.88 | 296.40 |
H3 | 65.3 | ng/mL | 13.06 | 391.80 |
A4 | 46.4 | ng/mL | 9.28 | 278.40 |
B4 | 63.6 | ng/mL | 12.72 | 381.60 |
C4 | 54.6 | ng/mL | 10.92 | 327.60 |
D4 | 48.5 | ng/mL | 9.70 | 291.00 |
E4 | 88.1 | ng/mL | 17.62 | 528.60 |
F4 | 51.4 | ng/mL | 10.28 | 308.40 |
G4 | 35.4 | ng/mL | 7.08 | 212.40 |
H4 | 66.3 | ng/mL | 13.26 | 397.80 |
A5 | 24.4 | ng/mL | 4.88 | 146.40 |
B5 | 24.3 | ng/mL | 4.86 | 145.80 |
C5 | 57.3 | ng/mL | 11.46 | 343.80 |
D5 | 59.2 | ng/mL | 11.84 | 355.20 |
E5 | 104 | ng/mL | 20.80 | 624.00 |
F5 | 55.8 | ng/mL | 11.16 | 334.80 |
G5 | 56.7 | ng/mL | 11.34 | 340.20 |
H5 | 73.2 | ng/mL | 14.64 | 439.20 |
B6 | 58 | ng/mL | 11.60 | 348.00 |
B7 | 55.8 | ng/mL | 11.16 | 334.80 |
D7 | 89.4 | ng/mL | 17.88 | 536.40 |
E7 | 30.7 | ng/mL | 6.14 | 184.20 |
G7 | 71 | ng/mL | 14.20 | 426.00 |
H7 | 26.8 | ng/mL | 5.36 | 160.80 |
A8 | 47.1 | ng/mL | 9.42 | 282.60 |
C8 | 36.4 | ng/mL | 7.28 | 218.40 |
D8 | 46.1 | ng/mL | 9.22 | 276.60 |
330k BSPP capture set up[edit]
Old probe ratio[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 |
DMR.s1 | 0.05 | 2.24 | 0.11 |
DMR.s2 | 0.07 | 2.24 | 0.16 |
DMR.s3 | 0.23 | 18.9 | 4.41 |
DMR.s4 | 1 | 18.9 | 18.9 |
DMR.s5 | 4.83 | 18.9 | 91.29 |
DMR.Exp1 | 0.23 | 18.9 | 4.44 |
DMR.Exp2 | 0.19 | 18.9 | 3.5 |
DMR.Exp3 | 0.27 | 18.9 | 5.03 |
- Dr. Zhang suggested to adjust probe ratio by increasing the ratio of the high performance ones which were used in very low ratio in the previous experiments
New probe ratio[edit]
S1 -S3 : increase 5x of the previous ratio S4: 1 (no change, as the base line) S5: no change Exp1-Exp3: increase to 1
Subset ID | Adjusted Multiplier F (2012_05_22) |
Actual amount of probe required (probe:target ratio, 100:1) |
Multiplier F x amount probe required |
Probe concentration (ng/ul) |
Volume (ul) | Volume for 75rxn (ul) | Subset ID |
DMR.s1 | 0.25 | 2.24 | 0.56 | 26.20 | 0.02 | 1.60 | DMR.s1 |
DMR.s2 | 0.35 | 2.24 | 0.78 | 30.10 | 0.03 | 1.95 | DMR.s2 |
DMR.s3 | 1.15 | 18.90 | 21.74 | 21.15 | 1.03 | 77.07 | DMR.s3 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 22.34 | 0.85 | 63.45 | DMR.s4 |
DMR.s5 | 4.83 | 18.90 | 91.29 | 29.00 | 3.15 | 236.09 | DMR.s5 |
DMR.Exp1 | 1.00 | 18.90 | 18.90 | 31.88 | 0.59 | 44.46 | DMR.Exp1 |
DMR.Exp2 | 1.00 | 18.90 | 18.90 | 24.11 | 0.78 | 58.79 | DMR.Exp2 |
DMR.Exp3 | 1.00 | 18.90 | 18.90 | 28.90 | 0.65 | 49.05 | DMR.Exp3 |
1.05uM Oligo suppressor-set1 | 0.125 | 9.375 | 1.05uM Oligo suppressor-set1 | ||||
1.05uM Oligo suppressor-set2 | 0.125 | 9.375 | 1.05uM Oligo suppressor-set2 |
- This means that the maximum volume of bis-cvt gDNA is 24.15ul in total volume 35ul
Components | 1rxn (ul) | 75 rxn mix (ul) |
10X AmpLigase Buffer | 3.50 | 262.50 |
DMR330k probe mix (ul) | 7.10 | 532.50 |
105nM Oligo suppressor | 0.25 | 18.75 |
Total volume (ul) | 10.85 | 813.75 |
- Aliquot 101 ul of reaction mix to striptube
Well IDs | Final conc. (ng/ul) | Volume for 250ng (ul) | Well IDs | H2O | Total amount of bis-cvt gDNA(ng) |
Probe mix (ul) | 10X AmpLigase Buffer (ul) | 1.05uM Oligo suppressor set1&2 (ul) |
Total volume (ul) |
A1 | 10.12 | 24.15 | A1 | 0.00 | 244.40 | 7.10 | 3.50 | 0.25 | 35.00 |
B1 | 9.58 | 24.15 | B1 | 0.00 | 231.36 | 7.10 | 3.50 | 0.25 | 35.00 |
C1 | 11.38 | 21.97 | C1 | 2.18 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
D1 | 12.64 | 19.78 | D1 | 4.37 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
E1 | 9.42 | 24.15 | E1 | 0.00 | 227.49 | 7.10 | 3.50 | 0.25 | 35.00 |
F1 | 13.24 | 18.88 | F1 | 5.27 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
G1 | 13.42 | 18.63 | G1 | 5.52 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
H1 | 11.94 | 20.94 | H1 | 3.21 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
A2 | 13.80 | 18.12 | A2 | 6.03 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
B2 | 10.64 | 23.50 | B2 | 0.65 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
C2 | 10.56 | 23.67 | C2 | 0.48 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
D2 | 11.14 | 22.44 | D2 | 1.71 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
E2 | 11.70 | 21.37 | E2 | 2.78 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
F2 | 12.18 | 20.53 | F2 | 3.62 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
G2 | 10.24 | 24.15 | G2 | 0.00 | 247.30 | 7.10 | 3.50 | 0.25 | 35.00 |
H2* | 7.36 | 24.15 | H2 | 0.00 | 177.74 | 7.10 | 3.50 | 0.25 | 35.00 |
A3 | 14.30 | 17.48 | A3 | 6.67 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
B3 | 11.50 | 21.74 | B3 | 2.41 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
C3 | 15.88 | 15.74 | C3 | 8.41 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
D3 | 8.84 | 24.15 | D3 | 0.00 | 213.49 | 7.10 | 3.50 | 0.25 | 35.00 |
E3 | 8.60 | 24.15 | E3 | 0.00 | 207.69 | 7.10 | 3.50 | 0.25 | 35.00 |
F3 | 11.62 | 21.51 | F3 | 2.64 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
G3 | 9.88 | 24.15 | G3 | 0.00 | 238.60 | 7.10 | 3.50 | 0.25 | 35.00 |
H3 | 13.06 | 19.14 | H3 | 5.01 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
A4 | 9.28 | 24.15 | A4 | 0.00 | 224.11 | 7.10 | 3.50 | 0.25 | 35.00 |
B4 | 12.72 | 19.65 | B4 | 4.50 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
C4 | 10.92 | 22.89 | C4 | 1.26 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
D4 | 9.70 | 24.15 | D4 | 0.00 | 234.26 | 7.10 | 3.50 | 0.25 | 35.00 |
E4 | 17.62 | 14.19 | E4 | 9.96 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
F4 | 10.28 | 24.15 | F4 | 0.00 | 248.26 | 7.10 | 3.50 | 0.25 | 35.00 |
G4* | 7.08 | 24.15 | G4 | 0.00 | 170.98 | 7.10 | 3.50 | 0.25 | 35.00 |
H4 | 13.26 | 18.85 | H4 | 5.30 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
A5* | 4.88 | 24.15 | A5 | 0.00 | 117.85 | 7.10 | 3.50 | 0.25 | 35.00 |
B5* | 4.86 | 24.15 | B5 | 0.00 | 117.37 | 7.10 | 3.50 | 0.25 | 35.00 |
C5 | 11.46 | 21.82 | C5 | 2.33 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
D5 | 11.84 | 21.11 | D5 | 3.04 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
E5 | 20.80 | 12.02 | E5 | 12.13 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
F5 | 11.16 | 22.40 | F5 | 1.75 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
G5 | 11.34 | 22.05 | G5 | 2.10 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
H5 | 14.64 | 17.08 | H5 | 7.07 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
A6 | 21.60 | 11.57 | A6 | 12.58 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
B6 | 11.60 | 21.55 | B6 | 2.60 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
C6 | 26.60 | 9.40 | C6 | 14.75 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
D6 | 28.00 | 8.93 | D6 | 15.22 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
E6 | 19.08 | 13.10 | E6 | 11.05 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
F6 | 27.00 | 9.26 | F6 | 14.89 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
G6 | 26.80 | 9.33 | G6 | 14.82 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
H6 | 19.22 | 13.01 | H6 | 11.14 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
A7 | 24.00 | 10.42 | A7 | 13.73 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
B7 | 11.16 | 22.40 | B7 | 1.75 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
C7 | 14.32 | 16.00 | C7 | 8.15 | 229.12 | 7.10 | 3.50 | 0.25 | 35.00 |
D7 | 17.88 | 13.98 | D7 | 10.17 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
E7* | 6.14 | 24.15 | E7 | 0.00 | 148.28 | 7.10 | 3.50 | 0.25 | 35.00 |
F7** | 1.43 | 16.00 | F7 | 8.15 | 22.82 | 7.10 | 3.50 | 0.25 | 35.00 |
G7 | 14.20 | 17.61 | G7 | 6.54 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
H7* | 5.36 | 24.15 | H7 | 0.00 | 129.44 | 7.10 | 3.50 | 0.25 | 35.00 |
A8 | 9.42 | 24.15 | A8 | 0.00 | 227.49 | 7.10 | 3.50 | 0.25 | 35.00 |
B8 | 26.20 | 9.54 | B8 | 14.61 | 250.00 | 7.10 | 3.50 | 0.25 | 35.00 |
C8* | 7.28 | 24.15 | C8 | 0.00 | 175.81 | 7.10 | 3.50 | 0.25 | 35.00 |
D8 | 9.22 | 24.15 | D8 | 0.00 | 222.66 | 7.10 | 3.50 | 0.25 | 35.00 |
E8** | 0.92 | 16.00 | E8 | 8.15 | 14.72 | 7.10 | 3.50 | 0.25 | 35.00 |
F8** | 1.48 | 16.00 | F8 | 8.15 | 23.68 | 7.10 | 3.50 | 0.25 | 35.00 |
- Note
- * Total amount of bis-cvt < 200ng
- ** Total amount of bis-cvt < 100ng
- 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): 101ul
- Aliquot reaction mix 10.85ul with multichannel pipette and mix well
- Put mineral oil in basin ~4 mL
- Layer the capture reaction with 40ul 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 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 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[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 240ul |
AmpliTaq Stoffel | 10 | U/ul | 2 | U/ul | 48.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 24.00 |
dNTP | 1000 | uM | 100 | uM | 24.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 24.00 |
H2O | 120.00 | ||||
Total | 240.00 |
- Aliquot 30ul of SLN mix to striptube
- Add 3ul of SLN mix to each well with multichannel pipettes
ExoI/III preparation[edit]
- Mix78 ul each of ExoI and ExoIII and aliquot 20ul into striptube
- Add 2ul to each well with multichannel pipettes
- Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-25