Noi/NOTES/2011-8-9: Difference between revisions

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* Aliquot 34.38 of SLN mix to strip tube and add 2.5ul to each well with multi-channel pipette
* Aliquot 34.38 of SLN mix to strip tube and add 2.5ul to each well with multi-channel pipette
= PCR amplification of captured DNA =
'''Plate1'''
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1rxn'''
| align="center" style="background:#f0f0f0;"|'''100 rxn mix'''
|-
| Captured template||10.00||0.00
|-
| 100uM AmpF6.4Sol||0.20||20.00
|-
| 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||37.00||3700.00
|-
| 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
* Since the first plate didn't give a very good amplification, I check back to my previous note and saw that the PCR profile that I currently used were a little different from the previous protocol. Then I changed the number of cycle in the first loop from 5x to 8x.
'''Plate2'''
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|''' Components'''
| align="center" style="background:#f0f0f0;"|'''1rxn'''
| align="center" style="background:#f0f0f0;"|'''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)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min
=== Plate layout and barcode primers ===
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''A1'''
| align="center" style="background:#f0f0f0;"|'''A2'''
| align="center" style="background:#f0f0f0;"|'''A3'''
| align="center" style="background:#f0f0f0;"|'''A4'''
| align="center" style="background:#f0f0f0;"|'''A5'''
| align="center" style="background:#f0f0f0;"|'''A6'''
| align="center" style="background:#f0f0f0;"|'''A7'''
| align="center" style="background:#f0f0f0;"|'''A8'''
| align="center" style="background:#f0f0f0;"|'''A9'''
| align="center" style="background:#f0f0f0;"|'''A10'''
| align="center" style="background:#f0f0f0;"|'''A11'''
| align="center" style="background:#f0f0f0;"|'''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
|}
= PAGE Quantification of Sequencing Libraries =
= PAGE Quantification of Sequencing Libraries =
* To purify the seq lib, 100ul of amplified amplicons plate2 were combined with 50ul of plate1 (total volume of amplified amplicon = 150ul) into 96-well plate
* To purify the seq lib, 100ul of amplified amplicons plate2 were combined with 50ul of plate1 (total volume of amplified amplicon = 150ul) into 96-well plate

Revision as of 18:04, 18 August 2011

Repeat BSPP DMR330k capture of GA and control gDNA from blood sample

  • 2011_08_08: received new plate of GA sample plate1 (so called GA plate1-2) from Ling. She prepared gDNA concentration 100ng/ul and volume ~10-15ul)
  • However, after measured the conc. by Nanodrop the conc. of the samples were very varied and were not enough for bisulfite conversion. The concentrations in plate 1-1 were verified by Nanodrop again. Then I calculated the volume required for 1200ng. I didn't pool the gDNA from the two plates because they have different conc. and some sample has too low conc.

Bisulfite conversion

  • Used EZ-96 DNA Methylation-Gold™ Kit (deep well format) Cat#D5008
  • Eluted volume 20ul with Elution buffer. After Qubit ss-DNA assay the total yields will be calculated based on ~16ul left in the well.
Sample ID Conc. (ng/ul) Volume plate1-2 enough? Volume plate1-2 (ul) Amount plate1-2 Conc. plate#1-1 (ng/ul) Volume plate 1-1 Sample ID H2O (ul) Total volume (ul) CT conversion reagent (ul) Total volume (ul)
A1 177.30 6.77 1200.00 A1 13.23 20.00 130.00 150.00
B1 104.91 11.44 1200.00 B1 8.56 20.00 130.00 150.00
C1 108.20 11.09 1200.00 C1 8.91 20.00 130.00 150.00
D1 95.30 12.59 1200.00 D1 7.41 20.00 130.00 150.00
E1 142.71 8.41 1200.00 E1 11.59 20.00 130.00 150.00
F1 130.28 9.21 1200.00 F1 10.79 20.00 130.00 150.00
G1 195.56 6.14 1200.00 G1 13.86 20.00 130.00 150.00
H1 126.34 9.50 1200.00 H1 10.50 20.00 130.00 150.00
A2 119.33 10.06 1200.00 A2 9.94 20.00 130.00 150.00
B2 135.56 8.85 1200.00 B2 11.15 20.00 130.00 150.00
C2 48.65 no 10.00 486.50 94.48 7.55 C2 2.45 20.00 130.00 150.00
D2 242.84 4.94 1200.00 D2 15.06 20.00 130.00 150.00
E2 109.93 10.92 1200.00 E2 9.08 20.00 130.00 150.00
F2 139.81 8.58 1200.00 F2 11.42 20.00 130.00 150.00
G2 87.91 13.65 1200.00 G2 6.35 20.00 130.00 150.00
H2 64.74 no 15.00 971.10 138.55 1.65 H2 3.35 20.00 130.00 150.00
A3 121.07 9.91 1200.00 A3 10.09 20.00 130.00 150.00
B3 103.64 11.58 1200.00 B3 8.42 20.00 130.00 150.00
C3 76.09 no 12.00 913.08 144.84 1.98 C3 6.02 20.00 130.00 150.00
D3 154.51 7.77 1200.00 D3 12.23 20.00 130.00 150.00
E3 198.30 6.05 1200.00 E3 13.95 20.00 130.00 150.00
F3 138.66 8.65 1200.00 F3 11.35 20.00 130.00 150.00
G3 163.20 7.35 1200.00 G3 12.65 20.00 130.00 150.00
H3 56.20 no 10.00 562.00 124.85 5.11 H3 4.89 20.00 130.00 150.00
A4 98.78 12.15 1200.00 A4 7.85 20.00 130.00 150.00
B4 60.47 no 12.00 725.64 151.88 3.12 B4 4.88 20.00 130.00 150.00
C4 66.43 no 12.00 797.16 119.93 3.36 C4 4.64 20.00 130.00 150.00
D4 119.64 10.03 1200.00 D4 9.97 20.00 130.00 150.00
E4 208.77 5.75 1200.00 E4 14.25 20.00 130.00 150.00
F4 83.48 no 12.00 1001.76 58.64 3.38 F4 4.62 20.00 130.00 150.00
G4 71.01 no 12.00 852.12 36.48 9.54 G4 0.00 21.54 130.00 151.54
H4 210.32 5.71 1200.00 H4 14.29 20.00 130.00 150.00
A5 266.74 4.50 1200.00 A5 15.50 20.00 130.00 150.00
B5 105.58 11.37 1200.00 B5 8.63 20.00 130.00 150.00
C5 89.41 13.42 1200.00 C5 6.58 20.00 130.00 150.00
D5 61.84 no 15.00 927.60 177.45 1.54 D5 3.46 20.00 130.00 150.00
E5 66.71 no 12.00 800.52 135.97 2.94 E5 5.06 20.00 130.00 150.00
F5 125.29 9.58 1200.00 F5 10.42 20.00 130.00 150.00
G5 121.31 9.89 1200.00 G5 10.11 20.00 130.00 150.00
H5 88.27 13.59 1200.00 H5 6.41 20.00 130.00 150.00
A6 134.70 8.91 1200.00 A6 11.09 20.00 130.00 150.00
B6 62.68 no 12.00 752.16 154.46 2.90 B6 5.10 20.00 130.00 150.00
C6 141.72 8.47 1200.00 C6 11.53 20.00 130.00 150.00
D6 187.04 6.42 1200.00 D6 13.58 20.00 130.00 150.00
E6 64.02 no 12.00 768.24 138.92 3.11 E6 4.89 20.00 130.00 150.00
F6 58.78 no 12.00 705.36 188.15 2.63 F6 5.37 20.00 130.00 150.00
G6 170.00 7.06 1200.00 G6 12.94 20.00 130.00 150.00
H6 111.00 10.81 1200.00 H6 9.19 20.00 130.00 150.00
A7 320.17 3.75 1200.00 A7 16.25 20.00 130.00 150.00
B7 162.36 7.39 1200.00 B7 12.61 20.00 130.00 150.00
C7 126.32 9.50 1200.00 C7 10.50 20.00 130.00 150.00
D7 368.67 3.25 1200.00 D7 16.75 20.00 130.00 150.00
E7 117.82 10.19 1200.00 E7 9.81 20.00 130.00 150.00
F7 84.38 no 12.00 1012.56 107.02 1.75 F7 6.25 20.00 130.00 150.00
G7 105.04 11.42 1200.00 G7 8.58 20.00 130.00 150.00
H7 86.61 13.86 1200.00 H7 6.14 20.00 130.00 150.00
A8 95.35 12.59 1200.00 A8 7.41 20.00 130.00 150.00
B8 98.32 12.21 1200.00 B8 7.79 20.00 130.00 150.00
C8 129.29 9.28 1200.00 C8 10.72 20.00 130.00 150.00
D8 100.98 11.88 1200.00 D8 8.12 20.00 130.00 150.00
E8 73.13 no 12.00 877.56 132.82 2.43 E8 5.57 20.00 130.00 150.00
F8 111.10 10.80 1200.00 F8 9.20 20.00 130.00 150.00
G8 30.00 no 10.00 300.00 162.10 5.55 G8 4.45 20.00 130.00 150.00
H8 82.39 no 12.00 988.68 182.77 1.16 H8 6.84 20.00 130.00 150.00
A9 61.85 no 12.00 742.20 136.13 3.36 A9 4.64 20.00 130.00 150.00
B9 110.49 10.86 1200.00 B9 9.14 20.00 130.00 150.00
C9 62.30 no 12.00 747.60 123.23 3.67 C9 4.33 20.00 130.00 150.00
D9 118.40 10.14 1200.00 D9 9.86 20.00 130.00 150.00
E9 75.01 16.00 1200.00 E9 4.00 20.00 130.00 150.00
F9 (tube 49.9) 60.63 no 15.00 909.45 38.24 7.60 F9 (tube 49.9) 0.00 22.60 130.00 152.60
G9 91.20 no 12.00 1094.40 142.40 0.74 G9 7.26 20.00 130.00 150.00
H9 90.62 13.24 1200.00 H9 6.76 20.00 130.00 150.00
A10 83.92 no 13.00 1090.96 193.87 0.56 A10 6.44 20.00 130.00 150.00
B10 120.56 9.95 1200.00 B10 10.05 20.00 130.00 150.00
C10 86.38 no 12.00 1036.56 168.48 0.97 C10 7.03 20.00 130.00 150.00
D10 49.85 no 12.00 598.20 172.60 3.49 D10 4.51 20.00 130.00 150.00
E10 123.63 9.71 1200.00 E10 10.29 20.00 130.00 150.00
F10 14.38 no 12.00 172.56 89.90 11.43 F10 0.00 23.43 130.00 153.43
G10 60.43 no 12.00 725.16 163.82 2.90 G10 5.10 20.00 130.00 150.00
H10 71.01 no 12.00 852.12 251.70 1.38 H10 6.62 20.00 130.00 150.00
A11 71.94 no 12.00 863.28 96.54 3.49 A11 4.51 20.00 130.00 150.00
B11 72.80 no 12.00 873.60 181.22 1.80 B11 6.20 20.00 130.00 150.00
C11 43.85 no 12.00 526.20 192.17 3.51 C11 4.49 20.00 130.00 150.00
D11 201.88 5.94 1200.00 D11 14.06 20.00 130.00 150.00
E11 221.63 5.41 1200.00 E11 14.59 20.00 130.00 150.00
F11 68.94 no 12.00 827.28 139.05 2.68 F11 5.32 20.00 130.00 150.00
G11 76.28 no 12.00 915.36 137.85 2.06 G11 5.94 20.00 130.00 150.00
H11 73.95 no 12.00 887.40 126.45 2.47 H11 5.53 20.00 130.00 150.00
A12 88.25 no 12.00 1059.00 126.33 1.12 A12 6.88 20.00 130.00 150.00
B12 (tube 94.9) 109.45 10.96 1200.00 B12 (tube 94.9) 9.04 20.00 130.00 150.00
C12 59.30 no 12.00 711.60 147.66 3.31 C12 4.69 20.00 130.00 150.00
D12 42.39 no 12.00 508.68 153.07 4.52 D12 3.48 20.00 130.00 150.00
E12 63.68 no 12.00 764.16 138.06 3.16 E12 4.84 20.00 130.00 150.00
F12 26.25 no 12.00 315.00 144.07 6.14 F12 1.86 20.00 130.00 150.00
G12 59.01 no 12.00 708.12 123.67 3.98 G12 4.02 20.00 130.00 150.00
H12 142.64 8.41 1200.00 H12 11.59 20.00 130.00 150.00


Qubit ssDNA assay

  • Follow protocol provided with the kit
  • Took 1ul of bis-cvt DNA into 9ul of H2O, then add the diluted DNA to 190ul ssDN Qubit reagent (dilution factor = 200)
Order Well ID Conc. in Qubit (ng/mL) Volume (ul) Final conc. (ng/ul) Yields (ng)
1.00 A1 217.00 16.00 43.40 694.40
2.00 B1 180.00 16.00 36.00 576.00
3.00 C1 204.00 16.00 40.80 652.80
4.00 D1 211.00 16.00 42.20 675.20
5.00 E1 201.00 16.00 40.20 643.20
6.00 F1 150.00 16.00 30.00 480.00
7.00 G1 135.00 16.00 27.00 432.00
8.00 H1 219.00 16.00 43.80 700.80
9.00 A2 190.00 16.00 38.00 608.00
10.00 B2 170.00 16.00 34.00 544.00
11.00 C2 151.00 16.00 30.20 483.20
12.00 D2 153.00 16.00 30.60 489.60
13.00 E2 292.00 16.00 58.40 934.40
14.00 F2 112.00 16.00 22.40 358.40
15.00 G2 160.00 16.00 32.00 512.00
16.00 H2 105.00 16.00 21.00 336.00
17.00 A3 176.00 16.00 35.20 563.20
18.00 B3 137.00 16.00 27.40 438.40
19.00 C3 119.00 16.00 23.80 380.80
20.00 D3 128.00 16.00 25.60 409.60
21.00 E3* 40.20 16.00 8.04 128.64
22.00 F3 134.00 16.00 26.80 428.80
23.00 G3 176.00 16.00 35.20 563.20
24.00 H3 112.00 16.00 22.40 358.40
25.00 A4 144.00 16.00 28.80 460.80
26.00 B4 135.00 16.00 27.00 432.00
27.00 C4 133.00 16.00 26.60 425.60
28.00 D4 139.00 16.00 27.80 444.80
29.00 E4 133.00 16.00 26.60 425.60
30.00 F4 189.00 16.00 37.80 604.80
31.00 G4 88.60 16.00 17.72 283.52
32.00 H4* 47.10 16.00 9.42 150.72
33.00 A5 148.00 16.00 29.60 473.60
34.00 B5 127.00 16.00 25.40 406.40
35.00 C5 83.70 16.00 16.74 267.84
36.00 D5 120.00 16.00 24.00 384.00
37.00 E5 117.00 16.00 23.40 374.40
38.00 F5* 34.40 16.00 6.88 110.08
39.00 G5 104.00 16.00 20.80 332.80
40.00 H5 112.00 16.00 22.40 358.40
41.00 A6 125.00 16.00 25.00 400.00
42.00 B6 126.00 16.00 25.20 403.20
43.00 C6 149.00 16.00 29.80 476.80
44.00 D6 135.00 16.00 27.00 432.00
45.00 E6 110.00 16.00 22.00 352.00
46.00 F6 102.00 16.00 20.40 326.40
47.00 G6 127.00 16.00 25.40 406.40
48.00 H6 59.40 16.00 11.88 190.08
49.00 A7 161.00 16.00 32.20 515.20
50.00 B7 146.00 16.00 29.20 467.20
51.00 C7 160.00 16.00 32.00 512.00
52.00 D7 135.00 16.00 27.00 432.00
53.00 E7 146.00 16.00 29.20 467.20
54.00 F7 133.00 16.00 26.60 425.60
55.00 G7 143.00 16.00 28.60 457.60
56.00 H7 85.20 16.00 17.04 272.64
57.00 A8 141.00 16.00 28.20 451.20
58.00 B8 147.00 16.00 29.40 470.40
59.00 C8 119.00 16.00 23.80 380.80
60.00 D8 81.40 16.00 16.28 260.48
61.00 E8 120.00 16.00 24.00 384.00
62.00 F8 62.20 16.00 12.44 199.04
63.00 G8 106.00 16.00 21.20 339.20
64.00 H8 135.00 16.00 27.00 432.00
65.00 A9 110.00 16.00 22.00 352.00
66.00 B9 147.00 16.00 29.40 470.40
67.00 C9 127.00 16.00 25.40 406.40
68.00 D9 95.70 16.00 19.14 306.24
69.00 E9 99.80 16.00 19.96 319.36
70.00 F9 104.00 16.00 20.80 332.80
71.00 G9 92.50 16.00 18.50 296.00
72.00 H9 116.00 16.00 23.20 371.20
73.00 A10 103.00 16.00 20.60 329.60
74.00 B10 69.70 16.00 13.94 223.04
75.00 C10 121.00 16.00 24.20 387.20
76.00 D10 141.00 16.00 28.20 451.20
77.00 E10 114.00 16.00 22.80 364.80
78.00 F10 154.00 16.00 30.80 492.80
79.00 G10 124.00 16.00 24.80 396.80
80.00 H10 111.00 16.00 22.20 355.20
81.00 A11 82.20 16.00 16.44 263.04
82.00 B11 112.00 16.00 22.40 358.40
83.00 C11 109.00 16.00 21.80 348.80
84.00 D11 61.50 16.00 12.30 196.80
85.00 E11 65.20 16.00 13.04 208.64
86.00 F11 148.00 16.00 29.60 473.60
87.00 G11 133.00 16.00 26.60 425.60
88.00 H11 200.00 16.00 40.00 640.00
89.00 A12 114.00 16.00 22.80 364.80
90.00 B12 149.00 16.00 29.80 476.80
91.00 C12 164.00 16.00 32.80 524.80
92.00 D12 142.00 16.00 28.40 454.40
93.00 E12 145.00 16.00 29.00 464.00
94.00 F12 137.00 16.00 27.40 438.40
95.00 G12 177.00 16.00 35.40 566.40
96.00 H12 202.00 16.00 40.40 646.40


  • Note: * = samples which total yields < 200ng

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 31.20 2.93 307.22
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


  • check volume of every well if they have the same level

Reaction mix

Components 1rxn (ul) 105 rxn mix (ul)
10X AmpLigase Buffer 2.50 262.50
DMR330k probe mix (ul) 4.53 475.65
105nM Oligo suppressor 0.30 31.50
Total volume (ul) 7.33 769.65


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 43.40 6.91 A1 10.76 300.00 4.53 2.50 0.30 25.00
B1 36.00 8.33 B1 9.34 300.00 4.53 2.50 0.30 25.00
C1 40.80 7.35 C1 10.32 300.00 4.53 2.50 0.30 25.00
D1 42.20 7.11 D1 10.56 300.00 4.53 2.50 0.30 25.00
E1 40.20 7.46 E1 10.21 300.00 4.53 2.50 0.30 25.00
F1 30.00 10.00 F1 7.67 300.00 4.53 2.50 0.30 25.00
G1 27.00 11.11 G1 6.56 300.00 4.53 2.50 0.30 25.00
H1 43.80 6.85 H1 10.82 300.00 4.53 2.50 0.30 25.00
A2 38.00 7.89 A2 9.78 300.00 4.53 2.50 0.30 25.00
B2 34.00 8.82 B2 8.85 300.00 4.53 2.50 0.30 25.00
C2 30.20 9.93 C2 7.74 300.00 4.53 2.50 0.30 25.00
D2 30.60 9.80 D2 7.87 300.00 4.53 2.50 0.30 25.00
E2 58.40 5.14 E2 12.53 300.00 4.53 2.50 0.30 25.00
F2 22.40 13.39 F2 4.28 300.00 4.53 2.50 0.30 25.00
G2 32.00 9.38 G2 8.30 300.00 4.53 2.50 0.30 25.00
H2 21.00 14.29 H2 3.38 300.00 4.53 2.50 0.30 25.00
A3 35.20 8.52 A3 9.15 300.00 4.53 2.50 0.30 25.00
B3 27.40 10.95 B3 6.72 300.00 4.53 2.50 0.30 25.00
C3 23.80 12.61 C3 5.06 300.00 4.53 2.50 0.30 25.00
D3 25.60 11.72 D3 5.95 300.00 4.53 2.50 0.30 25.00
E3* 8.04 16.00 E3* 1.67 128.64 4.53 2.50 0.30 25.00
F3 26.80 11.19 F3 6.48 300.00 4.53 2.50 0.30 25.00
G3 35.20 8.52 G3 9.15 300.00 4.53 2.50 0.30 25.00
H3 22.40 13.39 H3 4.28 300.00 4.53 2.50 0.30 25.00
A4 28.80 10.42 A4 7.25 300.00 4.53 2.50 0.30 25.00
B4 27.00 11.11 B4 6.56 300.00 4.53 2.50 0.30 25.00
C4 26.60 11.28 C4 6.39 300.00 4.53 2.50 0.30 25.00
D4 27.80 10.79 D4 6.88 300.00 4.53 2.50 0.30 25.00
E4 26.60 11.28 E4 6.39 300.00 4.53 2.50 0.30 25.00
F4 37.80 7.94 F4 9.73 300.00 4.53 2.50 0.30 25.00
G4 17.72 16.00 G4 1.67 283.52 4.53 2.50 0.30 25.00
H4* 9.42 16.00 H4* 1.67 150.72 4.53 2.50 0.30 25.00
A5 29.60 10.14 A5 7.53 300.00 4.53 2.50 0.30 25.00
B5 25.40 11.81 B5 5.86 300.00 4.53 2.50 0.30 25.00
C5 16.74 16.00 C5 1.67 267.84 4.53 2.50 0.30 25.00
D5 24.00 12.50 D5 5.17 300.00 4.53 2.50 0.30 25.00
E5 23.40 12.82 E5 4.85 300.00 4.53 2.50 0.30 25.00
F5* 6.88 16.00 F5* 1.67 110.08 4.53 2.50 0.30 25.00
G5 20.80 14.42 G5 3.25 300.00 4.53 2.50 0.30 25.00
H5 22.40 13.39 H5 4.28 300.00 4.53 2.50 0.30 25.00
A6 25.00 12.00 A6 5.67 300.00 4.53 2.50 0.30 25.00
B6 25.20 11.90 B6 5.77 300.00 4.53 2.50 0.30 25.00
C6 29.80 10.07 C6 7.60 300.00 4.53 2.50 0.30 25.00
D6 27.00 11.11 D6 6.56 300.00 4.53 2.50 0.30 25.00
E6 22.00 13.64 E6 4.03 300.00 4.53 2.50 0.30 25.00
F6 20.40 14.71 F6 2.96 300.00 4.53 2.50 0.30 25.00
G6 25.40 11.81 G6 5.86 300.00 4.53 2.50 0.30 25.00
H6 11.88 16.00 H6 1.67 190.08 4.53 2.50 0.30 25.00
A7 32.20 9.32 A7 8.35 300.00 4.53 2.50 0.30 25.00
B7 29.20 10.27 B7 7.40 300.00 4.53 2.50 0.30 25.00
C7 32.00 9.38 C7 8.30 300.00 4.53 2.50 0.30 25.00
D7 27.00 11.11 D7 6.56 300.00 4.53 2.50 0.30 25.00
E7 29.20 10.27 E7 7.40 300.00 4.53 2.50 0.30 25.00
F7 26.60 11.28 F7 6.39 300.00 4.53 2.50 0.30 25.00
G7 28.60 10.49 G7 7.18 300.00 4.53 2.50 0.30 25.00
H7 17.04 16.00 H7 1.67 272.64 4.53 2.50 0.30 25.00
A8 28.20 10.64 A8 7.03 300.00 4.53 2.50 0.30 25.00
B8 29.40 10.20 B8 7.47 300.00 4.53 2.50 0.30 25.00
C8 23.80 12.61 C8 5.06 300.00 4.53 2.50 0.30 25.00
D8 16.28 16.00 D8 1.67 260.48 4.53 2.50 0.30 25.00
E8 24.00 12.50 E8 5.17 300.00 4.53 2.50 0.30 25.00
F8 12.44 16.00 F8 1.67 199.04 4.53 2.50 0.30 25.00
G8 21.20 14.15 G8 3.52 300.00 4.53 2.50 0.30 25.00
H8 27.00 11.11 H8 6.56 300.00 4.53 2.50 0.30 25.00
A9 22.00 13.64 A9 4.03 300.00 4.53 2.50 0.30 25.00
B9 29.40 10.20 B9 7.47 300.00 4.53 2.50 0.30 25.00
C9 25.40 11.81 C9 5.86 300.00 4.53 2.50 0.30 25.00
D9 19.14 15.67 D9 2.00 300.00 4.53 2.50 0.30 25.00
E9 19.96 15.03 E9 2.64 300.00 4.53 2.50 0.30 25.00
F9 20.80 14.42 F9 3.25 300.00 4.53 2.50 0.30 25.00
G9 18.50 16.00 G9 1.67 296.00 4.53 2.50 0.30 25.00
H9 23.20 12.93 H9 4.74 300.00 4.53 2.50 0.30 25.00
A10 20.60 14.56 A10 3.11 300.00 4.53 2.50 0.30 25.00
B10 13.94 16.00 B10 1.67 223.04 4.53 2.50 0.30 25.00
C10 24.20 12.40 C10 5.27 300.00 4.53 2.50 0.30 25.00
D10 28.20 10.64 D10 7.03 300.00 4.53 2.50 0.30 25.00
E10 22.80 13.16 E10 4.51 300.00 4.53 2.50 0.30 25.00
F10 30.80 9.74 F10 7.93 300.00 4.53 2.50 0.30 25.00
G10 24.80 12.10 G10 5.57 300.00 4.53 2.50 0.30 25.00
H10 22.20 13.51 H10 4.16 300.00 4.53 2.50 0.30 25.00
A11 16.44 16.00 A11 1.67 263.04 4.53 2.50 0.30 25.00
B11 22.40 13.39 B11 4.28 300.00 4.53 2.50 0.30 25.00
C11 21.80 13.76 C11 3.91 300.00 4.53 2.50 0.30 25.00
D11 12.30 16.00 D11 1.67 196.80 4.53 2.50 0.30 25.00
E11 13.04 16.00 E11 1.67 208.64 4.53 2.50 0.30 25.00
F11 29.60 10.14 F11 7.53 300.00 4.53 2.50 0.30 25.00
G11 26.60 11.28 G11 6.39 300.00 4.53 2.50 0.30 25.00
H11 40.00 7.50 H11 10.17 300.00 4.53 2.50 0.30 25.00
A12 22.80 13.16 A12 4.51 300.00 4.53 2.50 0.30 25.00
B12 29.80 10.07 B12 7.60 300.00 4.53 2.50 0.30 25.00
C12 32.80 9.15 C12 8.52 300.00 4.53 2.50 0.30 25.00
D12 28.40 10.56 D12 7.11 300.00 4.53 2.50 0.30 25.00
E12 29.00 10.34 E12 7.33 300.00 4.53 2.50 0.30 25.00
F12 27.40 10.95 F12 6.72 300.00 4.53 2.50 0.30 25.00
G12 35.40 8.47 G12 9.20 300.00 4.53 2.50 0.30 25.00
H12 40.40 7.43 H12 10.24 300.00 4.53 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): 96.21 ul
  • Aliquot reaction mix 7.33 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
  • 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_14)
  -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_15)

SLN mix

  • Prepare 275 ul (2.5ul/rxn and prepare total for 110 rxn, more extra reaction to ensure that the volume enough when pipetting)
Components Stock conc. Unit Final conc. Unit Prepared 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 of SLN mix to strip tube and add 2.5ul to each well with multi-channel pipette

PCR amplification of captured DNA

Plate1

Components 1rxn 100 rxn mix
Captured template 10.00 0.00
100uM AmpF6.4Sol 0.20 20.00
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 37.00 3700.00
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

  • Since the first plate didn't give a very good amplification, I check back to my previous note and saw that the PCR profile that I currently used were a little different from the previous protocol. Then I changed the number of cycle in the first loop from 5x to 8x.

Plate2

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)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min

Plate layout and barcode primers

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

PAGE Quantification of Sequencing Libraries

  • To purify the seq lib, 100ul of amplified amplicons plate2 were combined with 50ul of plate1 (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 1ul of 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) Well ID conc. (ng/ul) Total amount in 60ul (ng) Volume for 25ng (ul)
A1 7.42 445.24 3.37 C1 5.62 337.48 4.44 E1 7.35 440.82 3.40
A2 5.64 338.19 4.44 C2 4.60 276.17 5.43 E2 6.74 404.16 3.71
A3 4.53 271.61 5.52 C3 3.07 184.27 8.14 E3** 0.19 11.46 60**
A4 4.10 245.97 6.10 C4 2.64 158.13 9.49 E4 3.41 204.74 7.33
A5 4.34 260.26 5.76 C5 0.95 56.71 26.45 E5 1.90 114.02 13.16
A6 4.37 262.10 5.72 C6 4.85 290.89 5.16 E6 1.36 81.30 18.45
A7 3.64 218.44 6.87 C7 4.26 255.41 5.87 E7 3.26 195.32 7.68
A8 9.62 577.08 2.60 C8 2.28 136.64 10.98 E8 2.49 149.17 10.06
A9 5.87 352.21 4.26 C9 0.65 39.17 38.30 E9 1.36 81.74 18.35
A10 7.38 442.57 3.39 C10 2.01 120.65 12.43 E10 2.20 132.04 11.36
B1 5.84 350.35 4.28 D1 5.56 333.37 4.50 F1 6.67 400.42 3.75
B2 5.46 327.84 4.58 D2 4.53 271.74 5.52 F2 3.02 181.16 8.28
B3 2.03 121.68 12.33 D3 1.88 112.91 13.28 F3 3.12 187.27 8.01
B4 2.88 172.65 8.69 D4 2.64 158.53 9.46 F4 2.70 162.27 9.24
B5 3.77 226.31 6.63 D5 1.90 114.20 13.14 F5** 0.10 6.08 60**
B6 3.43 205.99 7.28 D6 2.82 169.46 8.85 F6 2.18 130.98 11.45
B7 4.04 242.53 6.18 D7 2.95 177.20 8.47 F7 1.20 71.81 20.89
B8 3.92 235.42 6.37 D8** -0.45 -26.73 60** F8 0.73 43.78 34.26
B9 3.46 207.79 7.22 D9 1.52 91.15 16.46 F9 0.72 43.22 34.71
B10 0.58 34.94 42.93 D10 3.01 180.35 8.32 F10 4.79 287.50 5.22


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)
G1 5.54 332.34 4.51 A11 2.96 177.41 8.45
G2 4.10 245.73 6.10 B11 3.01 180.38 8.32
G3 3.70 222.02 6.76 C11 3.71 222.85 6.73
G4 0.89 53.24 28.17 D11 0.54 32.69 45.88
G5 1.61 96.85 15.49 E11 1.33 80.08 18.73
G6 3.74 224.69 6.68 F11 3.86 231.73 6.47
G7 2.78 166.71 9.00 G11 3.33 199.56 7.52
G8 1.18 70.72 21.21 H11 4.59 275.44 5.45
G9 0.80 48.13 31.16 A12 6.58 394.93 3.80
G10 3.28 196.77 7.62 B12 4.96 297.75 5.04
H1 5.58 334.76 4.48 C12 5.13 307.60 4.88
H2 3.56 213.89 7.01 D12 4.86 291.87 5.14
H3 4.43 266.06 5.64 E12 5.25 314.85 4.76
H4 1.13 67.98 22.07 F12 4.58 274.81 5.46
H5 2.94 176.59 8.49 G12 6.25 375.00 4.00
H6 3.21 192.76 7.78 H12 3.27 196.30 7.64
H7 1.73 103.53 14.49
H8 6.92 415.47 3.61
H9 2.34 140.57 10.67
H10 6.04 362.62 4.14


  • 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 ~ 100 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.