Noi/NOTES/2012-5-21

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DMR 330k BSPP capture of samples from Sergio[edit]

Repeat bisulfite conversion[edit]

Reagent prep
Add the following reagents to CT Conversion Reagent
  -  H2O                   9.0mL
  - M-Dissolving Buffer    500ul
  - M-Dilution Buffer      3.0mL
Mix at room temperature with frequent vortexing or shaking for 15 minutes
Add 144 ml of 100% ethanol to the 36 ml M-Wash Buffer concentrate before use
Lab IDs Conc. (ng/ul) Volume for 1200ng LabID ID Well IDs H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
Ap15-1 110.0 10.91 Ap15-1 A1 9.09 1200.00 130.00 150.00
Ap15-2 139.1 8.63 Ap15-2 B1 11.37 1200.00 130.00 150.00
Ap15-3 145.1 8.27 Ap15-3 C1 11.73 1200.00 130.00 150.00
Ap15-4 295.5 4.06 Ap15-4 D1 15.94 1200.00 130.00 150.00
Ap15-5 128.1 9.37 Ap15-5 E1 10.63 1200.00 130.00 150.00
Ap15-6 153.9 7.80 Ap15-6 F1 12.20 1200.00 130.00 150.00
Ap15-7 183.5 6.54 Ap15-7 G1 13.46 1200.00 130.00 150.00
Ap15-8 149.3 8.04 Ap15-8 H1 11.96 1200.00 130.00 150.00
Ap15-9 111.2 10.79 Ap15-9 A2 9.21 1200.00 130.00 150.00
Ap15-10 153.5 7.82 Ap15-10 B2 12.18 1200.00 130.00 150.00
Ap15-11 87.5 13.71 Ap15-11 C2 6.29 1200.00 130.00 150.00
Ap15-12 109.7 10.94 Ap15-12 D2 9.06 1200.00 130.00 150.00
Ap15-13 191.3 6.27 Ap15-13 E2 13.73 1200.00 130.00 150.00
Ap15-14 108.7 11.04 Ap15-14 F2 8.96 1200.00 130.00 150.00
Ap15-15 134.2 8.94 Ap15-15 G2 11.06 1200.00 130.00 150.00
Ap15-16 50.2 20.00 Ap15-16 H2 0.00 1200.00 130.00 150.00
Ap15-17 183.0 6.56 Ap15-17 A3 13.44 1200.00 130.00 150.00
Ap15-18 82.6 14.53 Ap15-18 B3 5.47 1200.00 130.00 150.00
Ap15-19 263.4 4.56 Ap15-19 C3 15.44 1200.00 130.00 150.00
Ma15-1 86.7 13.84 Ma15-1 D3 6.16 1200.00 130.00 150.00
Ma15-2 215.2 5.58 Ma15-2 E3 14.42 1200.00 130.00 150.00
Ma15-3 252.0 4.76 Ma15-3 F3 15.24 1200.00 130.00 150.00
Ma15-4 226.1 5.31 Ma15-4 G3 14.69 1200.00 130.00 150.00
Ma15-5 238.7 5.03 Ma15-5 H3 14.97 1200.00 130.00 150.00
Ma15-6 83.4 14.39 Ma15-6 A4 5.61 1200.00 130.00 150.00
Ma15-7 236.8 5.07 Ma15-7 B4 14.93 1200.00 130.00 150.00
Ma15-8 175.9 6.82 Ma15-8 C4 13.18 1200.00 130.00 150.00
Ma15-9 164.3 7.30 Ma15-9 D4 12.70 1200.00 130.00 150.00
Ma15-10 205.9 5.83 Ma15-10 E4 14.17 1200.00 130.00 150.00
Ma15-11 97.6 12.30 Ma15-11 F4 7.70 1200.00 130.00 150.00
Ma15-12 129.5 9.27 Ma15-12 G4 10.73 1200.00 130.00 150.00
Ma15-13 263.1 4.56 Ma15-13 H4 15.44 1200.00 130.00 150.00
Ma15-14 76.1 15.77 Ma15-14 A5 4.23 1200.00 130.00 150.00
Ma15-15 138.7 8.65 Ma15-15 B5 11.35 1200.00 130.00 150.00
Ma15-16 216.4 5.55 Ma15-16 C5 14.45 1200.00 130.00 150.00
Ma15-17 137.6 8.72 Ma15-17 D5 11.28 1200.00 130.00 150.00
Ma15-18 224.6 5.34 Ma15-18 E5 14.66 1200.00 130.00 150.00
Ma15-19 111.7 10.74 Ma15-19 F5 9.26 1200.00 130.00 150.00
Ma15-20 289.9 4.14 Ma15-20 G5 15.86 1200.00 130.00 150.00
Ma15-21 172.9 6.94 Ma15-21 H5 13.06 1200.00 130.00 150.00
Lab IDs Conc. (ng/ul) Volume for 1200ng LabID ID Well IDs H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
SR-2 90.8 13.22 SR-2 B6 6.78 1200.00 130.00 150.00
SR-10 184.8 6.49 SR-10 B7 13.51 1200.00 130.00 150.00
SR-11 105.4 5.00 SR-11 C7 15.00 527.00 130.00 150.00
SR-12 181.4 6.62 SR-12 D7 13.38 1200.00 130.00 150.00
SR-13 270.0 4.44 SR-13 E7 15.56 1200.00 130.00 150.00
SR-14 33.0 20.00 SR-14 F7 0.00 495.00 130.00 150.00
SR-15 67.0 17.91 SR-15 G7 2.09 1200.00 130.00 150.00
SR-16 145.6 8.24 SR-16 H7 11.76 1200.00 130.00 150.00
SR-17 245.3 4.89 SR-17 A8 15.11 1200.00 130.00 150.00
SR-19 159.5 7.52 SR-19 C8 12.48 1200.00 130.00 150.00
SR-20 195.7 6.13 SR-20 D8 13.87 1200.00 130.00 150.00
Well IDs/Sample IDs Well IDs/Sample IDs Well IDs/Sample IDs Well IDs/Sample IDs Well IDs/Sample IDs Well IDs/Sample IDs Well IDs/Sample IDs Well IDs/Sample IDs
A1 A2 A3 A4 A5 A6 A7 A8
Ap15-1 Ap15-9 Ap15-17 Ma15-6 Ma15-14 SR-1 => NO SR-9 SR-17
B1 B2 B3 B4 B5 B6 B7 B8
Ap15-2 Ap15-10 Ap15-18 Ma15-7 Ma15-15 SR-2 SR-10 SR-18 => NO
C1 C2 C3 C4 C5 C6 C7 C8
Ap15-3 Ap15-11 Ap15-19 Ma15-8 Ma15-16 SR-3 => NO SR-11 SR-19
D1 D2 D3 D4 D5 D6 D7 D8
Ap15-4 Ap15-12 Ma15-1 Ma15-9 Ma15-17 SR-4 => NO SR-12 SR-20
E1 E2 E3 E4 E5 E6 E7 E8
Ap15-5 Ap15-13 Ma15-2 Ma15-10 Ma15-18 SR-5 => NO SR-13 SR-21 => NO
F1 F2 F3 F4 F5 F6 F7 F8
Ap15-6 Ap15-14 Ma15-3 Ma15-11 Ma15-19 SR-6 => NO SR-14 SR-22 => NO
G1 G2 G3 G4 G5 G6 G7 G8
Ap15-7 Ap15-15 Ma15-4 Ma15-12 Ma15-20 SR-7 => NO SR-15
H1 H2 H3 H4 H5 H6 H7 H8
Ap15-8 Ap15-16 Ma15-5 Ma15-13 Ma15-21 SR-8 => NO SR-16
Note: no more gDNA for SR-14 samples
 Program
   98°C for 10 minutes
   64°C for 2.5 hours
   4°C storage up to 20 hours
Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA)

Qubit ssDNA assay[edit]

  • I combined bis-cvt gDNA of two batch together and measure concentration.
  • Sample in assay 1ul, dilution 200x
Sample Concentration in the Qubit ' Final conc. (ng/ul) Yields in 30ul (ng)
A1 50.6 ng/mL 10.12 303.60
B1 47.9 ng/mL 9.58 287.40
C1 56.9 ng/mL 11.38 341.40
D1 63.2 ng/mL 12.64 379.20
E1 47.1 ng/mL 9.42 282.60
F1 66.2 ng/mL 13.24 397.20
G1 67.1 ng/mL 13.42 402.60
H1 59.7 ng/mL 11.94 358.20
A2 69 ng/mL 13.80 414.00
B2 53.2 ng/mL 10.64 319.20
C2 52.8 ng/mL 10.56 316.80
D2 55.7 ng/mL 11.14 334.20
E2 58.5 ng/mL 11.70 351.00
F2 60.9 ng/mL 12.18 365.40
G2 51.2 ng/mL 10.24 307.20
H2 36.8 ng/mL 7.36 220.80
A3 71.5 ng/mL 14.30 429.00
B3 57.5 ng/mL 11.50 345.00
C3 79.4 ng/mL 15.88 476.40
D3 44.2 ng/mL 8.84 265.20
E3 43 ng/mL 8.60 258.00
F3 58.1 ng/mL 11.62 348.60
G3 49.4 ng/mL 9.88 296.40
H3 65.3 ng/mL 13.06 391.80
A4 46.4 ng/mL 9.28 278.40
B4 63.6 ng/mL 12.72 381.60
C4 54.6 ng/mL 10.92 327.60
D4 48.5 ng/mL 9.70 291.00
E4 88.1 ng/mL 17.62 528.60
F4 51.4 ng/mL 10.28 308.40
G4 35.4 ng/mL 7.08 212.40
H4 66.3 ng/mL 13.26 397.80
A5 24.4 ng/mL 4.88 146.40
B5 24.3 ng/mL 4.86 145.80
C5 57.3 ng/mL 11.46 343.80
D5 59.2 ng/mL 11.84 355.20
E5 104 ng/mL 20.80 624.00
F5 55.8 ng/mL 11.16 334.80
G5 56.7 ng/mL 11.34 340.20
H5 73.2 ng/mL 14.64 439.20
B6 58 ng/mL 11.60 348.00
B7 55.8 ng/mL 11.16 334.80
D7 89.4 ng/mL 17.88 536.40
E7 30.7 ng/mL 6.14 184.20
G7 71 ng/mL 14.20 426.00
H7 26.8 ng/mL 5.36 160.80
A8 47.1 ng/mL 9.42 282.60
C8 36.4 ng/mL 7.28 218.40
D8 46.1 ng/mL 9.22 276.60

330k BSPP capture set up[edit]

Old probe ratio[edit]

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
DMR.s1 0.05 2.24 0.11
DMR.s2 0.07 2.24 0.16
DMR.s3 0.23 18.9 4.41
DMR.s4 1 18.9 18.9
DMR.s5 4.83 18.9 91.29
DMR.Exp1 0.23 18.9 4.44
DMR.Exp2 0.19 18.9 3.5
DMR.Exp3 0.27 18.9 5.03
  • Dr. Zhang suggested to adjust probe ratio by increasing the ratio of the high performance ones which were used in very low ratio in the previous experiments

New probe ratio[edit]

S1 -S3 : increase 5x of the previous ratio
S4: 1 (no change, as the base line)
S5: no change
Exp1-Exp3: increase to 1
Subset ID Adjusted Multiplier F
(2012_05_22)
Actual amount of probe required
(probe:target ratio, 100:1)
Multiplier F x
amount probe required
Probe concentration
(ng/ul)
Volume (ul) Volume for 75rxn (ul) Subset ID
DMR.s1 0.25 2.24 0.56 26.20 0.02 1.60 DMR.s1
DMR.s2 0.35 2.24 0.78 30.10 0.03 1.95 DMR.s2
DMR.s3 1.15 18.90 21.74 21.15 1.03 77.07 DMR.s3
DMR.s4 1.00 18.90 18.90 22.34 0.85 63.45 DMR.s4
DMR.s5 4.83 18.90 91.29 29.00 3.15 236.09 DMR.s5
DMR.Exp1 1.00 18.90 18.90 31.88 0.59 44.46 DMR.Exp1
DMR.Exp2 1.00 18.90 18.90 24.11 0.78 58.79 DMR.Exp2
DMR.Exp3 1.00 18.90 18.90 28.90 0.65 49.05 DMR.Exp3
1.05uM Oligo suppressor-set1 0.125 9.375 1.05uM Oligo suppressor-set1
1.05uM Oligo suppressor-set2 0.125 9.375 1.05uM Oligo suppressor-set2
  • This means that the maximum volume of bis-cvt gDNA is 24.15ul in total volume 35ul
Components 1rxn (ul) 75 rxn mix (ul)
10X AmpLigase Buffer 3.50 262.50
DMR330k probe mix (ul) 7.10 532.50
105nM Oligo suppressor 0.25 18.75
Total volume (ul) 10.85 813.75
  • Aliquot 101 ul of reaction mix to striptube


Well IDs Final conc. (ng/ul) Volume for 250ng (ul) Well IDs H2O Total amount of
bis-cvt gDNA(ng)
Probe mix (ul) 10X AmpLigase Buffer (ul) 1.05uM Oligo suppressor
set1&2 (ul)
Total volume (ul)
A1 10.12 24.15 A1 0.00 244.40 7.10 3.50 0.25 35.00
B1 9.58 24.15 B1 0.00 231.36 7.10 3.50 0.25 35.00
C1 11.38 21.97 C1 2.18 250.00 7.10 3.50 0.25 35.00
D1 12.64 19.78 D1 4.37 250.00 7.10 3.50 0.25 35.00
E1 9.42 24.15 E1 0.00 227.49 7.10 3.50 0.25 35.00
F1 13.24 18.88 F1 5.27 250.00 7.10 3.50 0.25 35.00
G1 13.42 18.63 G1 5.52 250.00 7.10 3.50 0.25 35.00
H1 11.94 20.94 H1 3.21 250.00 7.10 3.50 0.25 35.00
A2 13.80 18.12 A2 6.03 250.00 7.10 3.50 0.25 35.00
B2 10.64 23.50 B2 0.65 250.00 7.10 3.50 0.25 35.00
C2 10.56 23.67 C2 0.48 250.00 7.10 3.50 0.25 35.00
D2 11.14 22.44 D2 1.71 250.00 7.10 3.50 0.25 35.00
E2 11.70 21.37 E2 2.78 250.00 7.10 3.50 0.25 35.00
F2 12.18 20.53 F2 3.62 250.00 7.10 3.50 0.25 35.00
G2 10.24 24.15 G2 0.00 247.30 7.10 3.50 0.25 35.00
H2* 7.36 24.15 H2 0.00 177.74 7.10 3.50 0.25 35.00
A3 14.30 17.48 A3 6.67 250.00 7.10 3.50 0.25 35.00
B3 11.50 21.74 B3 2.41 250.00 7.10 3.50 0.25 35.00
C3 15.88 15.74 C3 8.41 250.00 7.10 3.50 0.25 35.00
D3 8.84 24.15 D3 0.00 213.49 7.10 3.50 0.25 35.00
E3 8.60 24.15 E3 0.00 207.69 7.10 3.50 0.25 35.00
F3 11.62 21.51 F3 2.64 250.00 7.10 3.50 0.25 35.00
G3 9.88 24.15 G3 0.00 238.60 7.10 3.50 0.25 35.00
H3 13.06 19.14 H3 5.01 250.00 7.10 3.50 0.25 35.00
A4 9.28 24.15 A4 0.00 224.11 7.10 3.50 0.25 35.00
B4 12.72 19.65 B4 4.50 250.00 7.10 3.50 0.25 35.00
C4 10.92 22.89 C4 1.26 250.00 7.10 3.50 0.25 35.00
D4 9.70 24.15 D4 0.00 234.26 7.10 3.50 0.25 35.00
E4 17.62 14.19 E4 9.96 250.00 7.10 3.50 0.25 35.00
F4 10.28 24.15 F4 0.00 248.26 7.10 3.50 0.25 35.00
G4* 7.08 24.15 G4 0.00 170.98 7.10 3.50 0.25 35.00
H4 13.26 18.85 H4 5.30 250.00 7.10 3.50 0.25 35.00
A5* 4.88 24.15 A5 0.00 117.85 7.10 3.50 0.25 35.00
B5* 4.86 24.15 B5 0.00 117.37 7.10 3.50 0.25 35.00
C5 11.46 21.82 C5 2.33 250.00 7.10 3.50 0.25 35.00
D5 11.84 21.11 D5 3.04 250.00 7.10 3.50 0.25 35.00
E5 20.80 12.02 E5 12.13 250.00 7.10 3.50 0.25 35.00
F5 11.16 22.40 F5 1.75 250.00 7.10 3.50 0.25 35.00
G5 11.34 22.05 G5 2.10 250.00 7.10 3.50 0.25 35.00
H5 14.64 17.08 H5 7.07 250.00 7.10 3.50 0.25 35.00
A6 21.60 11.57 A6 12.58 250.00 7.10 3.50 0.25 35.00
B6 11.60 21.55 B6 2.60 250.00 7.10 3.50 0.25 35.00
C6 26.60 9.40 C6 14.75 250.00 7.10 3.50 0.25 35.00
D6 28.00 8.93 D6 15.22 250.00 7.10 3.50 0.25 35.00
E6 19.08 13.10 E6 11.05 250.00 7.10 3.50 0.25 35.00
F6 27.00 9.26 F6 14.89 250.00 7.10 3.50 0.25 35.00
G6 26.80 9.33 G6 14.82 250.00 7.10 3.50 0.25 35.00
H6 19.22 13.01 H6 11.14 250.00 7.10 3.50 0.25 35.00
A7 24.00 10.42 A7 13.73 250.00 7.10 3.50 0.25 35.00
B7 11.16 22.40 B7 1.75 250.00 7.10 3.50 0.25 35.00
C7 14.32 16.00 C7 8.15 229.12 7.10 3.50 0.25 35.00
D7 17.88 13.98 D7 10.17 250.00 7.10 3.50 0.25 35.00
E7* 6.14 24.15 E7 0.00 148.28 7.10 3.50 0.25 35.00
F7** 1.43 16.00 F7 8.15 22.82 7.10 3.50 0.25 35.00
G7 14.20 17.61 G7 6.54 250.00 7.10 3.50 0.25 35.00
H7* 5.36 24.15 H7 0.00 129.44 7.10 3.50 0.25 35.00
A8 9.42 24.15 A8 0.00 227.49 7.10 3.50 0.25 35.00
B8 26.20 9.54 B8 14.61 250.00 7.10 3.50 0.25 35.00
C8* 7.28 24.15 C8 0.00 175.81 7.10 3.50 0.25 35.00
D8 9.22 24.15 D8 0.00 222.66 7.10 3.50 0.25 35.00
E8** 0.92 16.00 E8 8.15 14.72 7.10 3.50 0.25 35.00
F8** 1.48 16.00 F8 8.15 23.68 7.10 3.50 0.25 35.00
  • Note
    • * Total amount of bis-cvt < 200ng
    • ** Total amount of bis-cvt < 100ng
  • 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): 101ul
  • Aliquot reaction mix 10.85ul with multichannel pipette and mix well
  • Put mineral oil in basin ~4 mL
  • Layer the capture reaction with 40ul 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 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) 
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 240ul
AmpliTaq Stoffel 10 U/ul 2 U/ul 48.00
AmpLigase 5 U/ul 0.5 U/ul 24.00
dNTP 1000 uM 100 uM 24.00
10x AmpLigase Buffer 10 x 1 x 24.00
H2O 120.00
Total 240.00
  • Aliquot 30ul of SLN mix to striptube
  • Add 3ul of SLN mix to each well with multichannel pipettes

ExoI/III preparation[edit]