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

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* TBE Gel:
* TBE Gel:
  [[File:HOTSPOTS_captureA_test.jpg | 300px]]
  [[File:HOTSPOTS_captureA_test.jpg | 300px]]
[[File:HOTSPOTS_captureA_test_repeat.job | 300px]]
  Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp.  
  Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp.  
  The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets.
  The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets.
* Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.
* Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.
* TBE Gel:
[[File:HOTSPOTS_captureA_E_newTemp.jpg | 300px]]

Revision as of 05:39, 27 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 kept for long period of times.
Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
1. 54 Ctrl H1 p54 Control TeSR 193.90 7.00 16.00 3.00 4.00 30
2. Act H1 p54 Activin-A 362.00 10.00 16.00 3.00 1.00 30
3. 47 Ctrl H1 p47 Control TeSR 144.95 6.50 16.00 3.00 4.50 30
4. 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

Capture gel

  • Amplified 4 uL in 25 uL reaction using KAPA 2xMM with AmpR6.3Ind17
 REAGENT                1x        5.5x
 Capture template     4.00 uL    --
 10 uM AmpF6.4Sol     0.50 uL   2.75 uL
 10 uM AmpF6.3RInd17  0.50 uL   2.75 uL
 2xKAPA Master Mix   12.50 uL  68.75 uL
 Nuclease free H2O    7.50 uL  41.25 uL
  • TBE Gel:
 File:HOTSPOTS capture1 test.jpg

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

Capture program

1. 95°C    30s
2. 94.8°C  30s
-0.2°C per 30s 174 times -> 60°C
3. Gradient: 55/56.4/58.8/60 °C    20h 
ADD 2.5 uL SLN mix
4. Gradient: 55/56.4/58.8/60 °C    20h 
5. 94°C    2m
ADD 2.0 uL ExoI/ExoIII mix
6. 37°C    2h
7. 90°C    5m
8. 4°C     forever

Capture PCR

  • 4 uL of capture was used to amplify. Two step PCR with 8 cycles first step, and 10 cycles second step = 18 cycles of PCR total for all samples.
  • TBE Gel:
File:HOTSPOTS captureA test.jpg
File:HOTSPOTS captureA test repeat.job
Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp. 
The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets.
  • Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.
  • TBE Gel:
File:HOTSPOTS captureA E newTemp.jpg