Dinh/Dinh 2014/NOTES/2014-3-11: Difference between revisions
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| Bmp||H1 p47 BMP-4||205.60||8.00||16.00||3.00||3.00||30 | | Bmp||H1 p47 BMP-4||205.60||8.00||16.00||3.00||3.00||30 | ||
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=== Capture program === | |||
1. 95°C 30s | |||
2. 94.8°C 30s | |||
-0.2°C per 30s 198 times | |||
3. 55°C 20h | |||
ADD 2.5 uL SLN mix | |||
4. 55°C 19.5h | |||
5. 94°C 2m | |||
ADD 2.0 uL ExoI/ExoIII mix | |||
6. 37°C 2h | |||
7. 90°C 5m | |||
8. 4°C forever | |||
== Test capture subset A == | == Test capture subset A == | ||
=== Capture setup === | === Capture setup === |
Revision as of 17:07, 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
- I used up all the bisulfite converted gDNA because the converted DNA cannot be keep long.
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 |
Capture program
1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 198 times 3. 55°C 20h ADD 2.5 uL SLN mix 4. 55°C 19.5h 5. 94°C 2m ADD 2.0 uL ExoI/ExoIII mix 6. 37°C 2h 7. 90°C 5m 8. 4°C forever
Test capture subset A
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 |
GM-A-1-55 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
GM-A-5-56.4 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
GM-A-8-58.8 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
GM-A-12-60 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |