Revision as of 19:42, 21 May 2015 by >Mzcai
in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set
Sample Groups
- gDNA 12878 (80.3ng/ul)
- Also a positive control and will be done using same volumes/amounts as all previous experiments
- cDNA dT BA8
- ~1/10th of gDNA amount
- cDNA N9 BA8
- ~1/10th of gDNA amount
- Negative Control
gDNA
Probe:target
|
1000:1
|
'
|
Probe size |
3514 |
probes
|
DNA templet |
300 |
ng
|
gDNA MW |
1.95x10^12 |
g/mol
|
gDNA (300ng) |
1.5385x10^-19 |
mol
|
Probe (1000:1) |
1.5385x10^-16 |
mol
|
Probe MW (3514, 150nt) |
1.71585106x10^8 |
g/mol
|
Amount Probe req'd |
26.4 |
ng
|
- 461nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 22.5 ng/ul
- Dilute to 16ul of 2.25ng/ul
- 10X less probes in cDNA than gDNA
- Make suppressor oligo pools with 5nM and 50nM of each supp oligos
- 1000-fold more of each suppressor oligo: 1.5385x10^-16 mole * 1000 = 1.5385x10^-13 mole = 1.5385x10^-4 nmole = 50 nM * 3.1ul
- 10X less probes in cDNA than gDNA
Sample #
|
Sample Description
|
Probes
|
Target
|
1000X Supp Oligos
|
10X Ampligase Buffer
|
H2O
|
Total
|
1 |
gDNA |
12 |
3.8 |
3.1 (50nM each) |
3 |
8 |
30
|
2 |
cDNA dT |
1.2 |
20 |
3.1 (5nM each) |
3 |
2.7 |
30
|
3 |
cDNA N9 |
1.2 |
20 |
3.1 (5nM each) |
3 |
2.7 |
30
|
4 |
NTC |
1.2 |
20 |
3.1 (5nM each) |
3 |
2.7 |
30
|
- Add 40ul Mineral Oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- In BioRad Thermalcycler program says: -0.2C per cycle every 30sec
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume 30ul
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
2.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
2.00
|
H2O |
|
|
|
|
16.00
|
Total |
|
|
|
|
20.00
|
Add Sequence Adapters PCR
Primers
Primer
|
Sequence
|
Index #
|
ISB_CA_AF |
AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG |
|
ISB_CA_AR.T1 |
CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG |
Indx1
|
ISB_CA_AR.T2 |
CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG |
Indx2
|
ISB_CA_AR.T3 |
CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG |
Indx3
|
Sample
|
Index
|
Forward Primer
|
Reverse Primer
|
1 |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
2 |
2 |
ISB_CA_AF |
ISB_CA_AR.T2
|
3 |
3 |
ISB_CA_AF |
ISB_CA_AR.T3
|
4 |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
PCR Test
Components
|
1X Volume
|
4.5X Volume
|
Captured template |
1 |
0
|
10uM Forward Primer |
0.4 |
1.8
|
10uM Reverse Primer |
0.4 |
0
|
2X KAPA SYBG MM |
12.5 |
56.25
|
H2O |
10.7 |
48.15
|
Total |
25 |
106.2
|
- Aliquot 23.6ul from 4.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min