Noi/NOTES/2011-8-14: Difference between revisions

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= Capture set up =
= Capture set up =
== Probe calculation ==
== Probe calculation ==
 
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Subset ID'''
| align="center" style="background:#f0f0f0;"|'''Adjusted Multiplier based on qPCR (Multiplier E)'''
| align="center" style="background:#f0f0f0;"|'''Actual amount of probe required (probe:target ratio, 100:1)'''
| align="center" style="background:#f0f0f0;"|'''Multiplier E x amount probe required'''
| align="center" style="background:#f0f0f0;"|'''Probe concentration (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''Volume for 105rxn (ul)'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| 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||||
|}<br>
== Reaction mix ==
== Reaction mix ==
 
{| {{table}} border = 1
 
| align="center" style="background:#f0f0f0;"|'''Components'''
 
| align="center" style="background:#f0f0f0;"|'''1rxn (ul)'''
| align="center" style="background:#f0f0f0;"|'''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
|}<br>
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''sample ID'''
| align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Volume for 300ng(ul)'''
| align="center" style="background:#f0f0f0;"|'''sample ID'''
| align="center" style="background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total amount of bis-cvt gDNA (ng)'''
| align="center" style="background:#f0f0f0;"|'''Probe mix (ul)'''
| align="center" style="background:#f0f0f0;"|'''10x Amligase buffer (ul)'''
| align="center" style="background:#f0f0f0;"|'''1.05uM Oligo suppressor set1&2 (ul)'''
| align="center" style="background:#f0f0f0;"|'''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
|}<br>
* Add H2O to the 96-well plate first then add bis-cvt gDNA. Note: Try to deliver reagents to the bottom of the well
* 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.
* 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): ..... ul
* Aliquot reaction mix in striptube (8 tubes): 81.77ul
* Aliquot reaction mix ...... ul with multichannel pipette and mix well
* Aliquot reaction mix 6.23ul with multichannel pipette and mix well
* Put mineral oil in basin ~3 mL)
* Put mineral oil in basin ~3 mL)
* Layer the capture reaction with 25ul mineral oil with multichannel pipette.
* Layer the capture reaction with 25ul mineral oil with multichannel pipette.

Revision as of 18:41, 15 August 2011

BSPP DMR330k capture of brain sample from Dr. Ophoff's lab (UCLA)

  • August 1, 2011, received 96 samples from Dr. Ophoff's lab
  • 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 adjust when perform bisulfite conversion
  • Note: The ratio of 260/230nm of most of the samples were very low, so I assumed high salt contamination.

Nanodrop measurement

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

  • 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

  • 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

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 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

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)