Noi/NOTES/2011-8-9

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Revision as of 17:21, 18 August 2011 by >Noi
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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