Dinh/Dinh 2014/NOTES/2014-3-11: Difference between revisions
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| F||100||97.4||9.74 | | F||100||97.4||9.74 | ||
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== Test capture 1 - 4 reactions == | |||
=== Pool subset probes for 4 reactions === | |||
{| class="wikitable" cellpadding=1 | |||
| align="center" style="background:#f0f0f0;"|'''Subset''' | |||
| align="center" style="background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="background:#f0f0f0;"|'''QUBIT(ng/mL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc(ng/uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume(uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount for 4.5 capture (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Subset Size''' | |||
|- | |||
| A||100||81.2||8.12||12.88||104.55||90000 | |||
|- | |||
| B||100||46.4||4.64||22.53||104.55||90000 | |||
|- | |||
| C||100||84.8||8.48||12.33||104.55||90000 | |||
|- | |||
| D||100||113||11.3||9.25||104.55||90000 | |||
|- | |||
| E||100||84.7||8.47||12.34||104.55||90000 | |||
|- | |||
| F||100||97.4||9.74||0.78||7.61||6553 | |||
|} | |||
* Pool probes for 4.5 captures and aliquot 15.58 uL per capture. | |||
=== Capture setup=== | |||
{| class="wikitable" cellpadding=1 | |||
| align="center" style="background:#f0f0f0;"|'''Tube ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Converted sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount sample (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume sample (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume probes (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume of 10x ampligase buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume water (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total volume''' | |||
|- | |||
| 54 Ctrl||H1 p54 Control TeSR||150.00||5.42||16.00||3.00||5.58||30 | |||
|- | |||
| Act||H1 p54 Activin-A||150.00||4.14||16.00||3.00||6.86||30 | |||
|- | |||
| 47 Ctrl||H1 p47 Control TeSR||150.00||6.73||16.00||3.00||4.27||30 | |||
|- | |||
| Bmp||H1 p47 BMP-4||150.00||5.84||16.00||3.00||5.16||30 | |||
|} | |} |
Revision as of 16:54, 19 March 2014
HOTSPOTS 460K Capture
Capture calculations
Size of Probes | 90000 | units |
gDNA | 150 | ng |
Human (MW) | 1.82E+012 | g/moles |
1 Probe (MW) | 31360.1 | g/moles |
Template | 8.23E-011 | nmoles |
Probes(100:1) | 8.23E-009 | nmoles |
Amount Probes needed | 23.23 | ng |
Size of Probes | 6553 | units |
gDNA | 150 | ng |
Human (MW) | 1.82E+012 | g/moles |
1 Probe (MW) | 31360.1 | g/moles |
Template | 8.23E-011 | nmoles |
Probes(100:1) | 8.23E-009 | nmoles |
Amount Probes needed | 1.69 | ng |
Human MW = (3x109 bp)*(607.4 gmoles-1/bp) + 157.9 gmoles-1 1 probe MW = (103 bp)*(303.7 gmoles-1)+ 79 gmoles-1 Amount probes = (1 probe MW) * (Size of probes) * (Moles of probes required)
Prepared probes
Subset | Dilution | QUBIT(ng/mL) | Conc(ng/uL) |
A | 100 | 81.2 | 8.12 |
B | 100 | 46.4 | 4.64 |
C | 100 | 84.8 | 8.48 |
D | 100 | 113 | 11.3 |
E | 100 | 84.7 | 8.47 |
F | 100 | 97.4 | 9.74 |
Test capture 1 - 4 reactions
Pool subset probes for 4 reactions
Subset | Dilution | QUBIT(ng/mL) | Conc(ng/uL) | Volume(uL) | Amount for 4.5 capture (ng) | Subset Size |
A | 100 | 81.2 | 8.12 | 12.88 | 104.55 | 90000 |
B | 100 | 46.4 | 4.64 | 22.53 | 104.55 | 90000 |
C | 100 | 84.8 | 8.48 | 12.33 | 104.55 | 90000 |
D | 100 | 113 | 11.3 | 9.25 | 104.55 | 90000 |
E | 100 | 84.7 | 8.47 | 12.34 | 104.55 | 90000 |
F | 100 | 97.4 | 9.74 | 0.78 | 7.61 | 6553 |
- Pool probes for 4.5 captures and aliquot 15.58 uL per capture.
Capture setup
Tube ID | Converted sample | Amount sample (ng) | Volume sample (uL) | Volume probes (uL) | Volume of 10x ampligase buffer | Volume water (uL) | Total volume |
54 Ctrl | H1 p54 Control TeSR | 150.00 | 5.42 | 16.00 | 3.00 | 5.58 | 30 |
Act | H1 p54 Activin-A | 150.00 | 4.14 | 16.00 | 3.00 | 6.86 | 30 |
47 Ctrl | H1 p47 Control TeSR | 150.00 | 6.73 | 16.00 | 3.00 | 4.27 | 30 |
Bmp | H1 p47 BMP-4 | 150.00 | 5.84 | 16.00 | 3.00 | 5.16 | 30 |