Noi/NOTES/2011-7-28

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BSPP DMR330k capture of GA and control gDNA from blood sample

Bisulfite conversion

  • Follow protocol provided with the kit.
  • EZ-96 DNA Methylation-Gold Kit
  • Catalog# D5007 (Shallow-Well Format)
  • Note:
    • Speed and time of centrifugation could be adjusted based on centrifuge model
    • Used 1 or1.2 ug of gDNA in bisulfite conversion reaction to get enough amount of bis-cvt DNA for BSPP capture. This experiment used 1.2ug
    • The minmum DNA concentration of gDNA is 50ng/ul.
    • Elute bis-cvt gDNA from the column in the last step with 20ul of Elution buffer to get the final volume ~ 17ul (will use 1 ul for Qubit ssDNA assay)

Qubit ss-DNA assay

  • Follow protocol provided with the kit
  • Add 1ul of bis-cvt gDNA sample to 199ul of 1x Qubit ssDNA reagent (total assay volume = 200ul)
Sample Conc. In Qubit (ng/mL) Volume (ul) Final conc (ng/ul) Yields (ng) Volume for 300ng Well ID
1.00 92.80 16.00 18.56 296.96 16.16 A1
2.00 704.00 16.00 140.80 2252.80 2.13 B1
3.00 84.00 16.00 49.40 790.40 6.07 C1*
4.00 109.00 16.00 21.80 348.80 13.76 D1
5.00 27.00 16.00 48.40 774.40 6.20 E1*
6.00 653.00 16.00 130.60 2089.60 2.30 F1
7.00 26.00 16.00 44.60 713.60 6.73 G1*
8.00 25.00 16.00 48.80 780.80 6.15 H1*
9.00 33.70 16.00 49.60 793.60 6.05 A2*
10.00 45.40 16.00 48.40 774.40 6.20 B2*
11.00 207.00 16.00 41.40 662.40 7.25 C2
12.00 200.00 16.00 40.00 640.00 7.50 D2
13.00 197.00 16.00 39.40 630.40 7.61 E2
14.00 115.00 16.00 23.00 368.00 13.04 F2
15.00 602.00 16.00 120.40 1926.40 2.49 G2
16.00 234.00 16.00 46.80 748.80 6.41 H2
17.00 39.70 16.00 46.40 742.40 6.47 A3*
18.00 104.00 16.00 20.80 332.80 14.42 B3
19.00 278.00 16.00 55.60 889.60 5.40 C3
20.00 109.00 16.00 21.80 348.80 13.76 D3
21.00 59.00 16.00 11.80 188.80 25.42 E3**
22.00 132.00 16.00 26.40 422.40 11.36 F3
23.00 12.50 16.00 44.60 713.60 6.73 G3*
24.00 207.00 16.00 41.40 662.40 7.25 H3
25.00 156.00 16.00 31.20 499.20 9.62 A4
26.00 174.00 16.00 34.80 556.80 8.62 B4
27.00 209.00 16.00 41.80 668.80 7.18 C4
28.00 75.60 16.00 42.40 678.40 7.08 D4*
29.00 156.00 16.00 31.20 499.20 9.62 E4
30.00 163.00 16.00 32.60 521.60 9.20 F4
31.00 220.00 16.00 44.00 704.00 6.82 G4
32.00 193.00 16.00 38.60 617.60 7.77 H4
33.00 440.00 16.00 88.00 1408.00 3.41 A5
34.00 252.00 16.00 50.40 806.40 5.95 B5
35.00 215.00 16.00 43.00 688.00 6.98 C5
36.00 188.00 16.00 37.60 601.60 7.98 D5
37.00 174.00 16.00 34.80 556.80 8.62 E5
38.00 152.00 16.00 30.40 486.40 9.87 F5
39.00 747.00 16.00 149.40 2390.40 2.01 G5
40.00 114.00 16.00 22.80 364.80 13.16 H5
41.00 540.00 16.00 108.00 1728.00 2.78 A6
42.00 258.00 16.00 51.60 825.60 5.81 B6
43.00 71.20 16.00 39.60 633.60 7.58 C6*
44.00 321.00 16.00 64.20 1027.20 4.67 D6
45.00 187.00 16.00 37.40 598.40 8.02 E6
46.00 167.00 16.00 33.40 534.40 8.98 F6
47.00 326.00 16.00 65.20 1043.20 4.60 G6
48.00 238.00 16.00 47.60 761.60 6.30 H6
49.00 408.00 16.00 81.60 1305.60 3.68 A7
50.00 141.00 16.00 28.20 451.20 10.64 B7
51.00 83.10 16.00 53.40 854.40 5.62 C7
52.00 265.00 16.00 53.00 848.00 5.66 D7
53.00 19.70 16.00 45.00 720.00 6.67 E7*
54.00 157.00 16.00 31.40 502.40 9.55 F7
55.00 63.10 16.00 44.80 716.80 6.70 G7*
56.00 177.00 16.00 35.40 566.40 8.47 H7
57.00 68.20 16.00 45.80 732.80 6.55 A8*
58.00 357.00 16.00 71.40 1142.40 4.20 B8
59.00 67.40 16.00 41.00 656.00 7.32 C8*
60.00 174.00 16.00 34.80 556.80 8.62 D8
61.00 197.00 16.00 39.40 630.40 7.61 E8
62.00 205.00 16.00 41.00 656.00 7.32 F8
63.00 226.00 16.00 45.20 723.20 6.64 G8
64.00 379.00 16.00 75.80 1212.80 3.96 H8
65.00 278.00 16.00 55.60 889.60 5.40 A9
66.00 359.00 16.00 71.80 1148.80 4.18 B9
67.00 156.00 16.00 31.20 499.20 9.62 C9
68.00 292.00 16.00 58.40 934.40 5.14 D9
69.00 216.00 16.00 43.20 691.20 6.94 E9
70.00 52.50 16.00 30.40 486.40 9.87 F9*
71.00 235.00 16.00 47.00 752.00 6.38 G9
72.00 237.00 16.00 47.40 758.40 6.33 H9
73.00 316.00 16.00 63.20 1011.20 4.75 A10
74.00 345.00 16.00 69.00 1104.00 4.35 B10
75.00 212.00 16.00 42.40 678.40 7.08 C10
76.00 263.00 16.00 52.60 841.60 5.70 D10
77.00 342.00 16.00 68.40 1094.40 4.39 E10
78.00 219.00 16.00 43.80 700.80 6.85 F10
79.00 220.00 16.00 44.00 704.00 6.82 G10
80.00 304.00 16.00 60.80 972.80 4.93 H10
81.00 119.00 16.00 23.80 380.80 12.61 A11
82.00 303.00 16.00 60.60 969.60 4.95 B11
83.00 247.00 16.00 49.40 790.40 6.07 C11
84.00 161.00 16.00 32.20 515.20 9.32 D11
85.00 143.00 16.00 28.60 457.60 10.49 E11
86.00 337.00 16.00 67.40 1078.40 4.45 F11
87.00 176.00 16.00 35.20 563.20 8.52 G11
88.00 315.00 16.00 63.00 1008.00 4.76 H11
89.00 232.00 16.00 46.40 742.40 6.47 A12
90.00 19.30 16.00 43.00 688.00 6.98 B12*
91.00 273.00 16.00 54.60 873.60 5.49 C12
92.00 257.00 16.00 51.40 822.40 5.84 D12
93.00 192.00 16.00 38.40 614.40 7.81 E12
94.00 195.00 16.00 39.00 624.00 7.69 F12
95.00 206.00 16.00 41.20 659.20 7.28 G12
96.00 316.00 16.00 63.20 1011.20 4.75 H12


  • repeat bisulfite conversion of samples with the yields < 300ng
Sample Conc. In Qubit (ng/mL) Volume (ul) Final conc (ng/ul) Yields (ng) Volume for 300ng Well ID
1.00 247.00 16.00 49.40 790.40 6.07 C1
2.00 242.00 16.00 48.40 774.40 6.20 E1
3.00 223.00 16.00 44.60 713.60 6.73 G1
4.00 244.00 16.00 48.80 780.80 6.15 H1
5.00 248.00 16.00 49.60 793.60 6.05 A2
6.00 242.00 16.00 48.40 774.40 6.20 B2
7.00 232.00 16.00 46.40 742.40 6.47 A3
8.00 55.50 16.00 11.10 177.60 27.03 E3
9.00 223.00 16.00 44.60 713.60 6.73 G3
10.00 212.00 16.00 42.40 678.40 7.08 D4
11.00 198.00 16.00 39.60 633.60 7.58 C6
12.00 267.00 16.00 53.40 854.40 5.62 C7
13.00 225.00 16.00 45.00 720.00 6.67 E7
14.00 224.00 16.00 44.80 716.80 6.70 G7
15.00 229.00 16.00 45.80 732.80 6.55 A8
16.00 205.00 16.00 41.00 656.00 7.32 C8
17.00 152.00 16.00 30.40 486.40 9.87 F9
18.00 215.00 16.00 43.00 688.00 6.98 B12


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 29.20 3.13 328.26
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
Total volume 5.03 528.46


Reaction mix

Components 1rxn (ul) 105rxn mix (ul)
10X AmpLigase Buffer 2.50 262.50
DMR330k probe mix (ul) 4.73 496.65
105nM Oligo suppressor 0.30 31.50
Total volume (ul) 7.53 790.65


sample ID Final conc. (ng/ul) Volume for 300ng(ul) 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 18.56 16.16 1.31 300.00 4.73 2.50 0.30 25.00
B1 140.80 2.13 15.34 300.00 4.73 2.50 0.30 25.00
C1* 49.40 6.07 11.40 300.00 4.73 2.50 0.30 25.00
D1 21.80 13.76 3.71 300.00 4.73 2.50 0.30 25.00
E1* 48.40 6.20 11.27 300.00 4.73 2.50 0.30 25.00
F1 130.60 2.30 15.17 300.00 4.73 2.50 0.30 25.00
G1* 44.60 6.73 10.74 300.00 4.73 2.50 0.30 25.00
H1* 48.80 6.15 11.32 300.00 4.73 2.50 0.30 25.00
A2* 49.60 6.05 11.42 300.00 4.73 2.50 0.30 25.00
B2* 48.40 6.20 11.27 300.00 4.73 2.50 0.30 25.00
C2 41.40 7.25 10.22 300.00 4.73 2.50 0.30 25.00
D2 40.00 7.50 9.97 300.00 4.73 2.50 0.30 25.00
E2 39.40 7.61 9.86 300.00 4.73 2.50 0.30 25.00
F2 23.00 13.04 4.43 300.00 4.73 2.50 0.30 25.00
G2 120.40 2.49 14.98 300.00 4.73 2.50 0.30 25.00
H2 46.80 6.41 11.06 300.00 4.73 2.50 0.30 25.00
A3* 46.40 6.47 11.00 300.00 4.73 2.50 0.30 25.00
B3 20.80 14.42 3.05 300.00 4.73 2.50 0.30 25.00
C3 55.60 5.40 12.07 300.00 4.73 2.50 0.30 25.00
D3 21.80 13.76 3.71 300.00 4.73 2.50 0.30 25.00
E3** 11.80 17.47 0 300.00 4.73 2.50 0.30 25.00
F3 26.40 11.36 6.11 300.00 4.73 2.50 0.30 25.00
G3* 44.60 6.73 10.74 300.00 4.73 2.50 0.30 25.00
H3 41.40 7.25 10.22 300.00 4.73 2.50 0.30 25.00
A4 31.20 9.62 7.85 300.00 4.73 2.50 0.30 25.00
B4 34.80 8.62 8.85 300.00 4.73 2.50 0.30 25.00
C4 41.80 7.18 10.29 300.00 4.73 2.50 0.30 25.00
D4* 42.40 7.08 10.39 300.00 4.73 2.50 0.30 25.00
E4 31.20 9.62 7.85 300.00 4.73 2.50 0.30 25.00
F4 32.60 9.20 8.27 300.00 4.73 2.50 0.30 25.00
G4 44.00 6.82 10.65 300.00 4.73 2.50 0.30 25.00
H4 38.60 7.77 9.70 300.00 4.73 2.50 0.30 25.00
A5 88.00 3.41 14.06 300.00 4.73 2.50 0.30 25.00
B5 50.40 5.95 11.52 300.00 4.73 2.50 0.30 25.00
C5 43.00 6.98 10.49 300.00 4.73 2.50 0.30 25.00
D5 37.60 7.98 9.49 300.00 4.73 2.50 0.30 25.00
E5 34.80 8.62 8.85 300.00 4.73 2.50 0.30 25.00
F5 30.40 9.87 7.60 300.00 4.73 2.50 0.30 25.00
G5 149.40 2.01 15.46 300.00 4.73 2.50 0.30 25.00
H5 22.80 13.16 4.31 300.00 4.73 2.50 0.30 25.00
A6 108.00 2.78 14.69 300.00 4.73 2.50 0.30 25.00
B6 51.60 5.81 11.66 300.00 4.73 2.50 0.30 25.00
C6* 39.60 7.58 9.89 300.00 4.73 2.50 0.30 25.00
D6 64.20 4.67 12.80 300.00 4.73 2.50 0.30 25.00
E6 37.40 8.02 9.45 300.00 4.73 2.50 0.30 25.00
F6 33.40 8.98 8.49 300.00 4.73 2.50 0.30 25.00
G6 65.20 4.60 12.87 300.00 4.73 2.50 0.30 25.00
H6 47.60 6.30 11.17 300.00 4.73 2.50 0.30 25.00
A7 81.60 3.68 13.79 300.00 4.73 2.50 0.30 25.00
B7 28.20 10.64 6.83 300.00 4.73 2.50 0.30 25.00
C7 53.40 5.62 11.85 300.00 4.73 2.50 0.30 25.00
D7 53.00 5.66 11.81 300.00 4.73 2.50 0.30 25.00
E7* 45.00 6.67 10.80 300.00 4.73 2.50 0.30 25.00
F7 31.40 9.55 7.92 300.00 4.73 2.50 0.30 25.00
G7* 44.80 6.70 10.77 300.00 4.73 2.50 0.30 25.00
H7 35.40 8.47 9.00 300.00 4.73 2.50 0.30 25.00
A8* 45.80 6.55 10.92 300.00 4.73 2.50 0.30 25.00
B8 71.40 4.20 13.27 300.00 4.73 2.50 0.30 25.00
C8* 41.00 7.32 10.15 300.00 4.73 2.50 0.30 25.00
D8 34.80 8.62 8.85 300.00 4.73 2.50 0.30 25.00
E8 39.40 7.61 9.86 300.00 4.73 2.50 0.30 25.00
F8 41.00 7.32 10.15 300.00 4.73 2.50 0.30 25.00
G8 45.20 6.64 10.83 300.00 4.73 2.50 0.30 25.00
H8 75.80 3.96 13.51 300.00 4.73 2.50 0.30 25.00
A9 55.60 5.40 12.07 300.00 4.73 2.50 0.30 25.00
B9 71.80 4.18 13.29 300.00 4.73 2.50 0.30 25.00
C9 31.20 9.62 7.85 300.00 4.73 2.50 0.30 25.00
D9 58.40 5.14 12.33 300.00 4.73 2.50 0.30 25.00
E9 43.20 6.94 10.53 300.00 4.73 2.50 0.30 25.00
F9* 30.40 9.87 7.60 300.00 4.73 2.50 0.30 25.00
G9 47.00 6.38 11.09 300.00 4.73 2.50 0.30 25.00
H9 47.40 6.33 11.14 300.00 4.73 2.50 0.30 25.00
A10 63.20 4.75 12.72 300.00 4.73 2.50 0.30 25.00
B10 69.00 4.35 13.12 300.00 4.73 2.50 0.30 25.00
C10 42.40 7.08 10.39 300.00 4.73 2.50 0.30 25.00
D10 52.60 5.70 11.77 300.00 4.73 2.50 0.30 25.00
E10 68.40 4.39 13.08 300.00 4.73 2.50 0.30 25.00
F10 43.80 6.85 10.62 300.00 4.73 2.50 0.30 25.00
G10 44.00 6.82 10.65 300.00 4.73 2.50 0.30 25.00
H10 60.80 4.93 12.54 300.00 4.73 2.50 0.30 25.00
A11 23.80 12.61 4.86 300.00 4.73 2.50 0.30 25.00
B11 60.60 4.95 12.52 300.00 4.73 2.50 0.30 25.00
C11 49.40 6.07 11.40 300.00 4.73 2.50 0.30 25.00
D11 32.20 9.32 8.15 300.00 4.73 2.50 0.30 25.00
E11 28.60 10.49 6.98 300.00 4.73 2.50 0.30 25.00
F11 67.40 4.45 13.02 300.00 4.73 2.50 0.30 25.00
G11 35.20 8.52 8.95 300.00 4.73 2.50 0.30 25.00
H11 63.00 4.76 12.71 300.00 4.73 2.50 0.30 25.00
A12 46.40 6.47 11.00 300.00 4.73 2.50 0.30 25.00
B12* 43.00 6.98 10.49 300.00 4.73 2.50 0.30 25.00
C12 54.60 5.49 11.98 300.00 4.73 2.50 0.30 25.00
D12 51.40 5.84 11.63 300.00 4.73 2.50 0.30 25.00
E12 38.40 7.81 9.66 300.00 4.73 2.50 0.30 25.00
F12 39.00 7.69 9.78 300.00 4.73 2.50 0.30 25.00
G12 41.20 7.28 10.19 300.00 4.73 2.50 0.30 25.00
H12 63.20 4.75 12.72 300.00 4.73 2.50 0.30 25.00


  • Note:
    • * = repeat bis-cvt samples
    • Sample E3 have total bis-cvt amount < 300ng
  • 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): 98.83 ul
  • Aliquot reaction mix 7.53 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
  • 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_07_29)
   -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_07_30)

SLN mix

Components Stock conc. Unit Final conc. Unit Prepared volume 220ul
AmpliTaq Stoffel 10.00 U/ul 2.00 U/ul 44.00
AmpLigase 5.00 U/ul 0.50 U/ul 22.00
dNTP 1000.00 uM 100.00 uM 22.00
10x AmpLigase Buffer 10.00 x 1.00 x 22.00
H2O 110.00
Total 220.00


  • SLN mix
    • Mix SLN mix in 1.5 mL tube
    • Aliquot 27.5 of SLN mix into striptube
    • Add 2 ul of SLN mix to each well with multi chennel pipette
  • Exo I/III mix: for 110 rxn mix (prepare ~ more rxn extra)
    • Mix 110ul each of ExoI and ExoIII in PCR tube and aliquot 27.5 ul into striptube
    • Add 2ul of ExoI/III to each capture reaction with multichannel pipette
  • Strictly suggestion: check the plate sealing every time to prevent sample evaporation

PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX)