Revision as of 22:44, 27 June 2016 by >Mzcai
in vitro Capture with TB12k_Apr2016 V7
- Will capture gDNA from mouse embryonic fibroblasts (got from Andrew)
Sample Groups
- gDNA MEF (77ng/ul)
- Negative Control
gDNA
Probe:target
|
1000:1
|
'
|
Probe size |
1808 |
probes
|
DNA template |
300 |
ng
|
gDNA MW |
1.8x10^12 |
g/mol
|
gDNA (300ng) |
1.66x10^-19 |
mol
|
Probe (1000:1) |
1.66x10^-16 |
mol
|
Probe MW (1808, 160nt) |
8.7997168x10^7 |
g/mol
|
Amount Probe req'd |
14.6 |
ng
|
Sample #
|
Sample Description
|
Probes
|
Target
|
10X Ampligase Buffer
|
H2O
|
Total
|
1 |
gDNA |
0.8 |
3.9 |
3 |
22.3 |
30
|
2 |
NTC |
0.8 |
0 |
3 |
26.2 |
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.
- Tiffany accidentally stopped reaction after 30min of Exo I/III digestion
- She let it run 2min at 94C and then put it in fridge
- Soon after, she called and I asked her to add 2ul of fresh Exo I/III and finish out the protocol with 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 |
1
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
1
|
H2O |
|
|
|
|
8
|
Total |
|
|
|
|
10
|
Add Sequence Adapters PCR
Primers
Primer
|
Sequence
|
Index #
|
ISB_CA_AF |
AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG |
|
ISB_CA_AR.T1 |
CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG |
Indx1
|
ISB_CA_AR.T2 |
CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG |
Indx2
|
Sample
|
Index
|
Forward Primer
|
Reverse Primer
|
gDNA |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
NTC |
2 |
ISB_CA_AF |
ISB_CA_AR.T2
|
PCR Test
Components
|
1X Volume
|
2.5X Volume
|
Captured template |
1 |
0
|
10uM Forward Primer |
0.4 |
1
|
10uM Reverse Primer |
0.4 |
0
|
2X KAPA SYBG MM |
12.5 |
31.25
|
H2O |
10.7 |
26.75
|
Total |
25 |
62.5
|
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21
450px