Revision as of 17:55, 22 June 2017 by >Mzcai
Agi15k_Feb2017 V4 + 5% formamide in vitro Capture
Calculate Probes Needed
V4
Probe:target
|
1000:1
|
'
|
Probe size |
4998 |
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 (4998, 157nt) |
2.387x10^8 |
g/mol
|
Amount Probe req'd |
36.7 |
ng
|
Probes, Target and Ampligase Buffer Mix
Sample
|
Condition
|
V4 (10.3ng/ul)
|
12878 DNA (80.3ng/ul)
|
UHRR (1ug/ul)
|
Ampligase Buffer
|
SplintR Buffer
|
Formamide or DMF
|
Ribolock 40U/ul
|
H2O
|
Total
|
1
|
Ampligase DNA
|
3.6
|
3.8
|
0
|
3
|
0
|
0
|
0
|
19.6
|
30
|
2
|
Ampligase NTC
|
3.6
|
0
|
0
|
3
|
0
|
0
|
0
|
23.4
|
30
|
3
|
SplintR 10% DMF DNA
|
3.6
|
3.8
|
0
|
0
|
3
|
3
|
0
|
16.6
|
30
|
4
|
SplintR 10% DMF RNA
|
3.6
|
0
|
1
|
0
|
3
|
3
|
0.5
|
18.9
|
30
|
5
|
SplintR 10% DMF NTC
|
3.6
|
0
|
0
|
0
|
3
|
3
|
0
|
20.4
|
30
|
6
|
SplintR 5% formamide DNA
|
3.6
|
3.8
|
0
|
0
|
3
|
1.5
|
0
|
18.1
|
30
|
7
|
SplintR 5% formamide RNA
|
3.6
|
0
|
1
|
0
|
3
|
1.5
|
0.5
|
20.4
|
30
|
8
|
SplintR 5% formamide NTC
|
3.6
|
0
|
0
|
0
|
3
|
1.5
|
0
|
21.9
|
30
|
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
- Should have added RiboLock after reached 55C since it would be denatured at 95C
- Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O)
- Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Take 30ul and purify with Zymo columns
- Left in 4C 1 hour after first column spin down
- Elute 10ul
AmpLigase enzyme mix
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume 10ul
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
1.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
1.00
|
H2O |
|
|
|
|
8.00
|
Total |
|
|
|
|
10.00
|
Quantify
- qPCR all 8 samples 1ul each with triplicates
Components
|
1X Volume
|
25X Volume
|
Captured template |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
10
|
10uM ISB_CA_AR.T2 |
0.4 |
10
|
2X KAPA SYBG MM |
12.5 |
312.5
|
H2O |
10.7 |
267.5
|
Total |
25 |
600
|
- Aliquot 24ul from 24X master mix and add 1ul captured template
Program
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min