Revision as of 03:24, 9 June 2017 by >Mzcai
Fifth Try: in tube SplintR Test with formamide
- Repeat Fourth Try with 95C 3min denature before hybridization
Test Conditions
- Standard: SplintR only
- SplintR + 5% formamide
- SplintR + 7.5% formamide
- SplintR + 10% formamide
- SplintR + 12.5% formamide
- SplintR + 15% formamide
- SplintR + 17.5% formamide
- Positive Control: Ampligase
- For each test conditions have
- one sample with ALL padlock probes and template
- one sample with all padlock probes with NO MALAT1 template
- Should not see amplification
Padlock Probes and Template
- ppCUX2
- ppBCL11B
- ppRELN_1
- ppGFAP
- ppMALAT1
- /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
- Template for ppMALAT1: MALAT1_template
- /5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT
Protocol
Sample #
|
Condition
|
30nM PP + Template
|
10X Buffer
|
Formamide
|
H2O
|
Total
|
1
|
SplintR
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
0
|
22.5 or 21.6
|
30
|
2
|
SplintR + 5% formamide
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
1.5
|
21 or 20.1
|
30
|
3
|
SplintR + 7.5% formamide
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
2.25
|
20.25 or 19.35
|
30
|
4
|
SplintR + 10% formamide
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
3
|
19.5 or 18.6
|
30
|
5
|
SplintR + 12.5% formamide
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
3.75
|
18.75 or 17.85
|
30
|
6
|
SplintR + 15% formamide
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
4.5
|
18 or 17.1
|
30
|
7
|
SplintR + 17.5 formamide
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
5.25
|
17.25 or 16.35
|
30
|
8
|
Ampligase
|
4.5 or 5.4 (0.9ul of each 1uM oligo)
|
3
|
0
|
22.5 or 21.6
|
30
|
- Combine padlock probes and template in 1X Ligase buffer and possibly formamide
- Add mineral oil on top
- Incubate at 95C for 3min
- Incubate at 55C for 18hr
- To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
- Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
- Move samples 1-7 to 37C
- Add 3ul SplintR Mix and incubate 15min
- SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O
- Incubate at 94C for 10min
- Put all samples on ice and add 2ul Exo I/III mix
- Incubate at 37C for 1hr
- Incubate at 94C for 10min
- qPCR all 16 samples with triplicates
Components
|
1X Volume
|
48X Volume
|
Captured template |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
19.2
|
10uM ISB_CA_AR.T2 |
0.4 |
19.2
|
2X KAPA SYBG MM |
12.5 |
600
|
H2O |
10.7 |
513.6
|
Total |
25 |
1,152
|
- Aliquot 24ul from 48X master mix and add 1ul captured template
Program
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
Results
raw data here
File:20170523 qPCR PPcapture SplintRformamide ScatterPlot.PNG
Repeat qPCR with 1/100 dilution
- For all 16 samples
- Do 2 serial dilutions of 1/10 to get 1/100
- 3ul + 27ul H2O -> 3ul + 27ul H2O
Components
|
1X Volume
|
48X Volume
|
Captured template diluted 1:100 |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
19.2
|
10uM ISB_CA_AR.T2 |
0.4 |
19.2
|
2X KAPA SYBG MM |
12.5 |
600
|
H2O |
10.7 |
513.6
|
Total |
25 |
1,152
|
- Aliquot 24ul from 48X master mix and add 1ul captured template
Program
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min