Noi/NOTES/2011-7-28: Difference between revisions
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* Pool ~25 ng of each amplified sampled into the same tube (=96X25 ng = 2.4ug) | * Pool ~25 ng of each amplified sampled into the same tube (=96X25 ng = 2.4ug) | ||
* Perform PAGE size selection in 6% TBE PAGE (use ~ 4 of 1mm 5-well gel to prevent overloading of DNA ) | * 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 as previously described | |||
* 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 by HiSeq. |
Revision as of 22:47, 29 July 2011
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 2ul 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)
- Spin the capture plate for 3 min to collect all liquid to the bottom of the well
- Use atleast 2/3 of the total volume for amplification (total volume =25ul +2+2 = 29ul).
- I alway use 8-10 ul in each PCR reaction by trying out the first PCR to see the qPCR curve and the number of cycle then perform the second PCR
Components | 1rxn | 100 rxn mix |
Captured template | 10.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 200.00 |
10uM AmpR6.3 (IndX, X 1,2,3,…,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)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
- Record the Ct value to compare the amplification of each sample
- The PCR can be stopped earlier depends on when it almost reach the plateau (do not over amplify).
Plate layout
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 |
- To purify the seq lib, 80ul of amplified amplicons were combined from two rounds (total volume of amplified amplicon = 160ul) into 96-well plate
- Purify seq lib with 0.8 volume Ampure beads, elute with 40ul EB
- Quantify DNA concentration of amplified amplicon in 6% TBE PAGE (12-well gel) (load 0.5ul of amplified amplicon)
- Pool ~25 ng of each amplified sampled into the same tube (=96X25 ng = 2.4ug)
- 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 as previously described
- 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 by HiSeq.