Noi/NOTES/2011-7-28: Difference between revisions

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(Created page with '= BSPP DMR330k capture of GA and control gDNA from blood sample = ==Qubit ss-DNA assay == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample''' | ali…')
 
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* repeat bisulfite conversion of samples with the yields < 300ng
* repeat bisulfite conversion of samples with the yields < 300ng
 
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Conc. In Qubit (ng/mL)'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''Final conc (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Yields (ng)'''
| align="center" style="background:#f0f0f0;"|'''Volume for 300ng'''
| align="center" style="background:#f0f0f0;"|'''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
|}<br>
== Capture set up ==
== Capture set up ==
=== Probe calculation ===
=== Probe calculation ===

Revision as of 20:52, 28 July 2011

BSPP DMR330k capture of GA and control gDNA from blood sample

Qubit ss-DNA assay

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 25.42 -7.95 300.00 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


  • 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,