Dinh/Dinh 2014/NOTES/2014-3-11: Difference between revisions

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=== Capture program ===
=== Capture program ===
  1. 95&degC   30s
  1. 95°C   30s
  2. 94.8&degC 30s
  2. 94.8°C 30s
  -0.2&degC per 30s 198 times
  -0.2°C per 30s 198 times
  3. 55&degC   20h  
  3. 55°C   20h  
  ADD 2.5 uL SLN mix
  ADD 2.5 uL SLN mix
  4. 55&degC   19.5h
  4. 55°C   19.5h
  5. 94&degC   2m
  5. 94°C   2m
  ADD 2.0 uL ExoI/ExoIII mix
  ADD 2.0 uL ExoI/ExoIII mix
  6. 37&degC   2h
  6. 37°C   2h
  7. 90&degC   5m
  7. 90°C   5m
  8. 4&degC     forever
  8. 4°C     forever
 
== Test capture subset A ==
== Test capture subset A ==
=== Capture setup ===
=== Capture setup ===

Revision as of 17:14, 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