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 | == Test capture - 4 reactions == | ||
=== Pool subset probes for 4 reactions === | === Pool subset probes for 4 reactions === | ||
{| class="wikitable" cellpadding=1 | {| class="wikitable" cellpadding=1 | ||
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| align="center" style="background:#f0f0f0;"|'''Total volume''' | | align="center" style="background:#f0f0f0;"|'''Total volume''' | ||
|- | |- | ||
| 54 Ctrl||H1 p54 Control TeSR|| | | 54 Ctrl||H1 p54 Control TeSR||193.90||7.00||16.00||3.00||4.00||30 | ||
|- | |- | ||
| Act||H1 p54 Activin-A|| | | Act||H1 p54 Activin-A||362.00||10.00||16.00||3.00||1.00||30 | ||
|- | |- | ||
| 47 Ctrl||H1 p47 Control TeSR|| | | 47 Ctrl||H1 p47 Control TeSR||144.95||6.50||16.00||3.00||4.50||30 | ||
|- | |- | ||
| Bmp||H1 p47 BMP-4|| | | Bmp||H1 p47 BMP-4||205.60||8.00||16.00||3.00||3.00||30 | ||
|} | |} |
Revision as of 16:58, 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 - 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 | 193.90 | 7.00 | 16.00 | 3.00 | 4.00 | 30 |
Act | H1 p54 Activin-A | 362.00 | 10.00 | 16.00 | 3.00 | 1.00 | 30 |
47 Ctrl | H1 p47 Control TeSR | 144.95 | 6.50 | 16.00 | 3.00 | 4.50 | 30 |
Bmp | H1 p47 BMP-4 | 205.60 | 8.00 | 16.00 | 3.00 | 3.00 | 30 |