Noi/NOTES/2011-7-28

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Revision as of 22:43, 29 July 2011 by >Noi
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BSPP DMR330k capture of GA and control gDNA from blood sample

Bisulfite conversion

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

Qubit ss-DNA assay

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


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


Capture set up

Probe calculation

Subset ID Adjusted Multiplier based on qPCR (Multiplier E) Actual amount of probe required (probe:target ratio, 100:1) Multiplier E x amount probe required Probe concentration (ng/ul) Volume (ul) Volume for 105rxn (ul)
DMR.s1 0.05 2.24 0.11 1.86 0.06 6.32
DMR.s2 0.07 2.24 0.16 1.95 0.08 8.77
DMR.s3 0.23 18.90 4.41 23.20 0.19 19.94
DMR.s4 1.00 18.90 18.90 23.20 0.81 85.54
DMR.s5** 4.83 18.90 91.29 29.20 3.13 328.26
DMR.Exp1 0.23 18.90 4.44 31.88 0.14 14.61
DMR.Exp2 0.19 18.90 3.50 24.11 0.15 15.26
DMR.Exp3 0.27 18.90 5.03 28.90 0.17 18.26
1.05uM Oligo suppressor-set1 0.15 15.75
1.05uM Oligo suppressor-set2 0.15 15.75
Total volume 5.03 528.46


Reaction mix

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


sample ID Final conc. (ng/ul) Volume for 300ng(ul) H2O (ul) Total amount of bis-cvt gDNA (ng) Probe mix (ul) 10x Amligase buffer (ul) 1.05uM Oligo suppressor set1&2 (ul) Total volume (ul)
A1 18.56 16.16 1.31 300.00 4.73 2.50 0.30 25.00
B1 140.80 2.13 15.34 300.00 4.73 2.50 0.30 25.00
C1* 49.40 6.07 11.40 300.00 4.73 2.50 0.30 25.00
D1 21.80 13.76 3.71 300.00 4.73 2.50 0.30 25.00
E1* 48.40 6.20 11.27 300.00 4.73 2.50 0.30 25.00
F1 130.60 2.30 15.17 300.00 4.73 2.50 0.30 25.00
G1* 44.60 6.73 10.74 300.00 4.73 2.50 0.30 25.00
H1* 48.80 6.15 11.32 300.00 4.73 2.50 0.30 25.00
A2* 49.60 6.05 11.42 300.00 4.73 2.50 0.30 25.00
B2* 48.40 6.20 11.27 300.00 4.73 2.50 0.30 25.00
C2 41.40 7.25 10.22 300.00 4.73 2.50 0.30 25.00
D2 40.00 7.50 9.97 300.00 4.73 2.50 0.30 25.00
E2 39.40 7.61 9.86 300.00 4.73 2.50 0.30 25.00
F2 23.00 13.04 4.43 300.00 4.73 2.50 0.30 25.00
G2 120.40 2.49 14.98 300.00 4.73 2.50 0.30 25.00
H2 46.80 6.41 11.06 300.00 4.73 2.50 0.30 25.00
A3* 46.40 6.47 11.00 300.00 4.73 2.50 0.30 25.00
B3 20.80 14.42 3.05 300.00 4.73 2.50 0.30 25.00
C3 55.60 5.40 12.07 300.00 4.73 2.50 0.30 25.00
D3 21.80 13.76 3.71 300.00 4.73 2.50 0.30 25.00
E3** 11.80 17.47 0 300.00 4.73 2.50 0.30 25.00
F3 26.40 11.36 6.11 300.00 4.73 2.50 0.30 25.00
G3* 44.60 6.73 10.74 300.00 4.73 2.50 0.30 25.00
H3 41.40 7.25 10.22 300.00 4.73 2.50 0.30 25.00
A4 31.20 9.62 7.85 300.00 4.73 2.50 0.30 25.00
B4 34.80 8.62 8.85 300.00 4.73 2.50 0.30 25.00
C4 41.80 7.18 10.29 300.00 4.73 2.50 0.30 25.00
D4* 42.40 7.08 10.39 300.00 4.73 2.50 0.30 25.00
E4 31.20 9.62 7.85 300.00 4.73 2.50 0.30 25.00
F4 32.60 9.20 8.27 300.00 4.73 2.50 0.30 25.00
G4 44.00 6.82 10.65 300.00 4.73 2.50 0.30 25.00
H4 38.60 7.77 9.70 300.00 4.73 2.50 0.30 25.00
A5 88.00 3.41 14.06 300.00 4.73 2.50 0.30 25.00
B5 50.40 5.95 11.52 300.00 4.73 2.50 0.30 25.00
C5 43.00 6.98 10.49 300.00 4.73 2.50 0.30 25.00
D5 37.60 7.98 9.49 300.00 4.73 2.50 0.30 25.00
E5 34.80 8.62 8.85 300.00 4.73 2.50 0.30 25.00
F5 30.40 9.87 7.60 300.00 4.73 2.50 0.30 25.00
G5 149.40 2.01 15.46 300.00 4.73 2.50 0.30 25.00
H5 22.80 13.16 4.31 300.00 4.73 2.50 0.30 25.00
A6 108.00 2.78 14.69 300.00 4.73 2.50 0.30 25.00
B6 51.60 5.81 11.66 300.00 4.73 2.50 0.30 25.00
C6* 39.60 7.58 9.89 300.00 4.73 2.50 0.30 25.00
D6 64.20 4.67 12.80 300.00 4.73 2.50 0.30 25.00
E6 37.40 8.02 9.45 300.00 4.73 2.50 0.30 25.00
F6 33.40 8.98 8.49 300.00 4.73 2.50 0.30 25.00
G6 65.20 4.60 12.87 300.00 4.73 2.50 0.30 25.00
H6 47.60 6.30 11.17 300.00 4.73 2.50 0.30 25.00
A7 81.60 3.68 13.79 300.00 4.73 2.50 0.30 25.00
B7 28.20 10.64 6.83 300.00 4.73 2.50 0.30 25.00
C7 53.40 5.62 11.85 300.00 4.73 2.50 0.30 25.00
D7 53.00 5.66 11.81 300.00 4.73 2.50 0.30 25.00
E7* 45.00 6.67 10.80 300.00 4.73 2.50 0.30 25.00
F7 31.40 9.55 7.92 300.00 4.73 2.50 0.30 25.00
G7* 44.80 6.70 10.77 300.00 4.73 2.50 0.30 25.00
H7 35.40 8.47 9.00 300.00 4.73 2.50 0.30 25.00
A8* 45.80 6.55 10.92 300.00 4.73 2.50 0.30 25.00
B8 71.40 4.20 13.27 300.00 4.73 2.50 0.30 25.00
C8* 41.00 7.32 10.15 300.00 4.73 2.50 0.30 25.00
D8 34.80 8.62 8.85 300.00 4.73 2.50 0.30 25.00
E8 39.40 7.61 9.86 300.00 4.73 2.50 0.30 25.00
F8 41.00 7.32 10.15 300.00 4.73 2.50 0.30 25.00
G8 45.20 6.64 10.83 300.00 4.73 2.50 0.30 25.00
H8 75.80 3.96 13.51 300.00 4.73 2.50 0.30 25.00
A9 55.60 5.40 12.07 300.00 4.73 2.50 0.30 25.00
B9 71.80 4.18 13.29 300.00 4.73 2.50 0.30 25.00
C9 31.20 9.62 7.85 300.00 4.73 2.50 0.30 25.00
D9 58.40 5.14 12.33 300.00 4.73 2.50 0.30 25.00
E9 43.20 6.94 10.53 300.00 4.73 2.50 0.30 25.00
F9* 30.40 9.87 7.60 300.00 4.73 2.50 0.30 25.00
G9 47.00 6.38 11.09 300.00 4.73 2.50 0.30 25.00
H9 47.40 6.33 11.14 300.00 4.73 2.50 0.30 25.00
A10 63.20 4.75 12.72 300.00 4.73 2.50 0.30 25.00
B10 69.00 4.35 13.12 300.00 4.73 2.50 0.30 25.00
C10 42.40 7.08 10.39 300.00 4.73 2.50 0.30 25.00
D10 52.60 5.70 11.77 300.00 4.73 2.50 0.30 25.00
E10 68.40 4.39 13.08 300.00 4.73 2.50 0.30 25.00
F10 43.80 6.85 10.62 300.00 4.73 2.50 0.30 25.00
G10 44.00 6.82 10.65 300.00 4.73 2.50 0.30 25.00
H10 60.80 4.93 12.54 300.00 4.73 2.50 0.30 25.00
A11 23.80 12.61 4.86 300.00 4.73 2.50 0.30 25.00
B11 60.60 4.95 12.52 300.00 4.73 2.50 0.30 25.00
C11 49.40 6.07 11.40 300.00 4.73 2.50 0.30 25.00
D11 32.20 9.32 8.15 300.00 4.73 2.50 0.30 25.00
E11 28.60 10.49 6.98 300.00 4.73 2.50 0.30 25.00
F11 67.40 4.45 13.02 300.00 4.73 2.50 0.30 25.00
G11 35.20 8.52 8.95 300.00 4.73 2.50 0.30 25.00
H11 63.00 4.76 12.71 300.00 4.73 2.50 0.30 25.00
A12 46.40 6.47 11.00 300.00 4.73 2.50 0.30 25.00
B12* 43.00 6.98 10.49 300.00 4.73 2.50 0.30 25.00
C12 54.60 5.49 11.98 300.00 4.73 2.50 0.30 25.00
D12 51.40 5.84 11.63 300.00 4.73 2.50 0.30 25.00
E12 38.40 7.81 9.66 300.00 4.73 2.50 0.30 25.00
F12 39.00 7.69 9.78 300.00 4.73 2.50 0.30 25.00
G12 41.20 7.28 10.19 300.00 4.73 2.50 0.30 25.00
H12 63.20 4.75 12.72 300.00 4.73 2.50 0.30 25.00


  • Note:
    • * = repeat bis-cvt samples
    • Sample E3 have total bis-cvt amount < 300ng
  • Add H2O to the 96-well plate first then add bis-cvt gDNA. Note: Try to deliver reagents to the bottom of the well
  • Prepare reaction mix in 1.5 mL tube by pipetting probe, oligo suppressor and 10x AmLigase buffer and mix well.
  • Aliquot reaction mix in striptube (8 tubes): 98.83 ul
  • Aliquot reaction mix 7.53 ul with multichannel pipette and mix well
  • Put mineral oil in basin ~3 mL)
  • Layer the capture reaction with 20ul mineral oil with multichannel pipette.
  • Seal the plate with microseal and make sure that every well is completely sealed to prevent evaporation
  • Centrifuge the capture plate for 3 min and place the plate on PCR machine with the following profile,

Program

   -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
   -> add 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 )