Noi/NOTES/2011-8-14: Difference between revisions
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* 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 adjust when perform 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 adjust when perform bisulfite conversion | ||
* Note: The ratio of 260/230nm of most of the samples were very low, so I assumed | * Note: The ratio of 260/230nm of most of the samples were very low, so I assumed high salt contamination. | ||
== Nanodrop measurement == | == Nanodrop measurement == | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
Line 251: | Line 251: | ||
| H12||74.57||16.09||H12||3.91||1200.00||130.00||150.00 | | H12||74.57||16.09||H12||3.91||1200.00||130.00||150.00 | ||
|} | |} | ||
* Eluted volume | * 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: | * Note: | ||
** *= Total amount in bis-cvt lower than 1200ng, but still above 1000ng | ** *= 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) | ** ** 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 | ** ***= 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)<br> | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Well ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit (ng/mL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|}<br> | |||
= Capture set up = | |||
== Probe calculation == | |||
== Reaction mix == | |||
* 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): ..... ul | |||
* Aliquot reaction mix ...... ul 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) |
Revision as of 18:09, 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
Reaction mix
- 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): ..... ul
- Aliquot reaction mix ...... ul 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)