Revision as of 06:57, 21 March 2014 by >Dinh
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
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
File:HOTSPOTS captureA test.jpg