Revision as of 00:34, 9 April 2017 by >Mzcai
SplintR RNA template specificity
Purpose
Samples
- PP + gDNA + SplintR
- PP + SplintR
- PP -ppCUX2 + SplintR
- PP (negative control for qPCR)
- PP + CircLigase II (positive control)
Protocol
- SplintR reaction for Samples 1-4
- Incubate at 25C for 20min
- Heat inactivate at 65C for 20min
Sample
|
10X SplintR Buffer
|
SplintR Ligase
|
10uM ppBCL11B
|
10uM ppCUX2
|
10uM ppGFAP
|
10uM ppRELN_1
|
gDNA 12878 80.3ng/ul
|
H2O
|
Total
|
1
|
3
|
3
|
0.3
|
0.3
|
0.3
|
0.3
|
3
|
19.8
|
30
|
2
|
3
|
3
|
0.3
|
0.3
|
0.3
|
0.3
|
0
|
22.8
|
30
|
3
|
3
|
3
|
0.3
|
0
|
0.3
|
0.3
|
0
|
23.1
|
30
|
4
|
0
|
0
|
0.3
|
0.3
|
0.3
|
0.3
|
0
|
28.8
|
30
|
- Circligase II reaction with 400nM total
- Incubate at 60C for 1hr
- Inactive at 80C for 10min
Components
|
Volume
|
CircLigase II 10X Buffer
|
3
|
CircLigase II Enzyme
|
1.5
|
5M Betaine
|
6
|
50mM MnCl2
|
1.5
|
10uM ppBCL11B
|
0.3
|
10uM ppCUX2
|
0.3
|
10uM ppGFAP
|
0.3
|
10uM ppRELN_1
|
0.3
|
H2O
|
16.8
|
Total
|
30
|
- Split each sample into 2 15ul samples
- Exo I/III digest one of each sample by adding 1ul Exo I and 1ul Exo III
- Incubate at 37C for 1.5 hour
- Purify all 10 samples with Zymo ssDNA/RNA columns
- qPCR all 10 samples
Components
|
1X Volume
|
16X Volume
|
Captured template |
5 |
0
|
10uM ISB_CA_AF |
0.4 |
6.4
|
10uM ISB_CA_AR.T1 |
0.4 |
6.4
|
2X KAPA SYBG MM |
12.5 |
200
|
H2O |
6.7 |
107.2
|
Total |
25 |
320
|
- Aliquot 20ul from 16X master mix and add 5ul captured template
Program
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
- Run gel on both