Noi/NOTES/2011-8-14: Difference between revisions
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= BSPP DMR330k capture of blood sample from Dr. Ophoff's lab (UCLA) = | = 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 | * August 1, 2011, received 96 gDNA of the blood samples from Dr. Ophoff's lab. They are the blood sample of schizophrenia disorder patients. | ||
* Tina Wang confirmed the concentration of each sample was about 50ng/ul | * 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 | * 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 | ||
Line 796: | Line 796: | ||
'''Program'''<br> | '''Program'''<br> | ||
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 (stop at 11 cycles) -> 72C 3min<br> | 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 (stop at 11 cycles) -> 72C 3min<br> | ||
=== Plate layout and | === Plate layout and barcoded primer === | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''A1''' | | align="center" style="background:#f0f0f0;"|'''A1''' | ||
Line 963: | Line 963: | ||
* 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 in 6 tubes | * Purify DNA from acrylamide gel in 6 tubes | ||
=== PAGE size-selection === | === PAGE size-selection === | ||
* Flush the well with 1 mL pipette to remove residual gel | * Flush the well with 1 mL pipette to remove residual gel |
Latest revision as of 00:31, 21 September 2011
BSPP DMR330k capture of blood sample from Dr. Ophoff's lab (UCLA)[edit]
- August 1, 2011, received 96 gDNA of the blood samples from Dr. Ophoff's lab. They are the blood sample of schizophrenia disorder patients.
- 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[edit]
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[edit]
- 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[edit]
- 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[edit]
Probe calculation[edit]
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[edit]
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[edit]
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[edit]
- 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)[edit]
- 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 barcoded primer[edit]
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[edit]
- 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.