Noi/NOTES/2011-8-14

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BSPP DMR330k capture of blood sample from Dr. Ophoff's lab (UCLA)

  • August 1, 2011, received 96 gDNA of the blood 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 adjusted when performed 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) (ng) Multiplier E x amount probe required (ng) 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 stripp tube
  • Add 2.5ul of SLN mix 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 5ul 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 (stop at 11 cycles) -> 72C 3min

Plate layout and barcode primer

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


  • Repeated PCR with the same condition
  • To purify the seq lib, 75ul of amplified amplicons were combined from two rounds (total volume of amplified amplicon = 150ul) into 96-well plate
  • Purify seq lib with 0.7 volume Ampure beads, elute with 60ul EB
  • Quantify DNA concentration of amplified amplicon in 6% TBE PAGE (12-well gel) (load 1 ul of purified amplified amplicon)
  • Pool ~25 ng of each amplified sampled into the same tube (=96X25 ng = 2.4ug)
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 5.17 310.47 4.83 E1 3.24 194.46 7.71
A2 5.11 306.56 4.89 E2 3.20 191.84 7.82
A3 2.57 154.12 9.73 E3 2.91 174.53 8.59
A4 7.33 439.77 3.41 E4 4.35 260.90 5.75
A5 4.49 269.29 5.57 E5 6.11 366.54 4.09
A6 7.92 475.32 3.16 E6 4.72 283.17 5.30
A7 5.62 336.91 4.45 E7 4.97 297.90 5.04
A8 5.31 318.46 4.71 E8 7.80 468.20 3.20
A9 9.85 591.15 2.54 E9 5.30 318.18 4.71
A10 6.27 376.04 3.99 E10 6.95 417.28 3.59
B1 6.57 394.02 3.81 F1 3.70 222.28 6.75
B2 4.25 254.99 5.88 F2 4.38 262.51 5.71
B3 4.66 279.73 5.36 F3 2.53 151.81 9.88
B4 3.74 224.12 6.69 F4 2.82 169.24 8.86
B5 4.99 299.42 5.01 F5 4.69 281.50 5.33
B6 5.96 357.80 4.19 F6 3.86 231.78 6.47
B7 5.34 320.57 4.68 F7 4.37 261.93 5.73
B8 4.79 287.62 5.22 F8 2.85 170.88 8.78
B9 7.40 443.85 3.38 F9 4.90 293.83 5.11
B10 4.98 298.90 5.02 F10 2.59 155.52 9.65
C1 2.93 175.55 8.54 G1 5.03 301.72 4.97
C2 5.40 323.77 4.63 G2 2.76 165.69 9.05
C3 1.99 119.40 12.56 G3 4.38 262.54 5.71
C4 3.68 221.05 6.79 G4 3.38 202.76 7.40
C5 1.90 114.19 13.14 G5 4.90 294.26 5.10
C6 4.98 298.58 5.02 G6 4.46 267.72 5.60
C7 3.51 210.56 7.12 G7 6.57 394.26 3.80
C8 5.26 315.78 4.75 G8 7.48 448.57 3.34
C9 5.21 312.51 4.80 G9 7.90 474.09 3.16
C10 5.73 343.90 4.36 G10 6.50 389.71 3.85
D1 5.08 304.92 4.92 H1 6.38 382.51 3.92
D2 3.22 193.37 7.76 H2 4.78 286.92 5.23
D3 2.90 173.77 8.63 H3 5.89 353.41 4.24
D4 4.82 289.30 5.18 H4 5.75 344.87 4.35
D5 3.74 224.35 6.69 H5 8.38 502.81 2.98
D6 3.65 219.01 6.85 H6 4.80 287.94 5.21
D7 5.16 309.33 4.85 H7 5.91 354.78 4.23
D8 2.37 141.94 10.57 H8 7.83 469.93 3.19
D9 8.19 491.18 3.05 H9 6.54 392.26 3.82
D10 11.57 694.15 2.16 H10 2.46 147.51 10.17
A11 6.28 376.93 3.98 A12 9.04 542.64 2.76
B11 5.55 332.72 4.51 B12 6.39 383.13 3.92
C11 4.42 264.91 5.66 C12 6.22 373.21 4.02
D11 7.02 421.27 3.56 D12 5.38 322.69 4.65
E11 6.07 363.97 4.12 E12 11.83 709.77 2.11
F11 5.68 340.88 4.40 F12 9.17 550.36 2.73
G11 6.24 374.68 4.00 G12 10.73 643.59 2.33
H11 7.59 455.21 3.30 H12 10.34 620.25 2.42
  • 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 in 6 tubes

PAGE size-selection

  • Flush the well with 1 mL pipette to remove residual gel
  • Load the library mix in each well and load 25bp ladder (Invitrogen) in the middle well.
  • Run at 200 volts for 30 min in 0.5X TBE buffer.
  • Remove the gel from the cassette and stain in ~ 20 mL 0.5XTBE buffer with 1X SYBR Gold (add 2 ul SYBR Gold to 20 mL 0.5X TBE buffer)
  • Stain for 3 min on the shaker.
  • Place stained gel on a clean Saran wrap on the UV box with the long wavelength.
  • Locate the position approx. 375 bp according to 25bp DNA ladder. Use scalpel to cut the gel as close as to the band and minimize exposing the gel to UV light.
  • Move the gel slide to the a clean area and chop the gel piece to small pieces with scalpel and transfer to prepared tube (one gel go to two tubes or more)
  • Close the lid and centrifuge the tube at 15,000 rpm, for 3 min at RT.
  • Transfer the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
  • Add 500 ul of 1X TE buffer.
  • Shake vigorously on vortex for 45-60 min at 37 C.
  • Centrifuge at 15,000 rpm for 2 min at RT.
  • Transfer the clear spnt. to Nanosep column and centrifuge at 15,000 rpm for 1 min.
  • Transfer the lower layer to Nanosep again, but try to avoid the large chunk of gel. Centrifuge with the same speed about 3min.
  • Transfer spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
  • Precipitate with 2.5 volume of 100% EtOH, 1/300 volume of Glycoblue, 0.1 volume of 3M NaoAc pH 5.2.
  • Mix well and place the tube at -80C for at least 15 min.
  • Centrifuge 10,000 rpm for 20 min at 4C.
  • Discard spnt and wash with 800 ul 75% cold EtOH, spin at 14,000rpm for 5min at room temp centrifuge.
  • Dry the DNA pellet and dissolve with ~20 ul H2O/tube.
  • Pool the dissolved probe to the same tube and mix well.
  • Estimate DNA concentration using 6% TBE gel (12-well, 1mm cassette)

Tubes preparation for PAGE size selection

  • Place 0.5 mL tube into 1.5 mL DNA LoBind tube.
  • Punch 0.5 mL tube with 22G needle
  • Treat the tube under UV light for 15 min if possible
  • 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.