Revision as of 23:19, 18 May 2017 by >Mzcai
Agi15k_Feb2017 V4 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
|
H2O
|
Total
|
1
|
Ampligase DNA
|
3.6
|
3.8
|
0
|
3
|
0
|
0
|
19.6
|
30
|
2
|
Ampligase NTC
|
3.6
|
0
|
0
|
3
|
0
|
0
|
23.4
|
30
|
3
|
SplintR DNA
|
3.6
|
3.8
|
0
|
0
|
3
|
0
|
19.6
|
30
|
4
|
SplintR RNA
|
3.6
|
0
|
1
|
0
|
3
|
0
|
22.4
|
30
|
5
|
SplintR NTC
|
3.6
|
0
|
0
|
0
|
3
|
0
|
23.4
|
30
|
6
|
SplintR 20% formamide DNA
|
3.6
|
3.8
|
0
|
0
|
3
|
6
|
13.6
|
30
|
7
|
SplintR 20% formamide RNA
|
3.6
|
0
|
1
|
0
|
3
|
6
|
16.4
|
30
|
8
|
SplintR 20% formamide NTC
|
3.6
|
0
|
0
|
0
|
3
|
6
|
17.4
|
30
|
Program
- 1,2,3,6: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
- 4,5,7,8: 55C 18h
- Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- SplintR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
- Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- SplintR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Zymo columns
- Elute 10ul
- Sample 2 was only able to pipette 20ul instead of 30ul of the ligation reaction so theoretically has 33% lower conc after elution from column
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