Matt:LabNotes/2017-5-8
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Second Try: in tube SplintR Test with formamide and ET SSB
- First try showed SplintR with 10% formamide had the best sensitivity and specificity of the conditions tried
- Will try varying formamide concentration around 10%
- Will qPCR with lower starting conc to get more accurate measurement
Test Conditions
- Standard: SplintR only
- SplintR + 5% formamide
- SplintR + 7.5% formamide
- SplintR + 10% formamide
- SplintR + 12.5% formamide
- SplintR + 15% formamide
- SplintR + 10% formamide + 250ng ET SSB
- Positive Control: Ampligase
- For each test conditions have
- one sample with ALL padlock probes and template
- Should see amplification
- one sample with all padlock probes with NO MALAT1 template
- Should not see amplification
- one sample with ALL padlock probes and template
Padlock Probes and Template
- ppCUX2
- ppBCL11B
- ppRELN_1
- ppGFAP
- ppMALAT1
- /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
- Template for ppMALAT1: MALAT1_template
- /5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT
Protocol
Sample # | Condition | 100nM PP + Template | 10X Buffer | Formamide | H2O | Total |
1 | SplintR | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 0 | 25.5 or 25.2 | 30 |
2 | SplintR + 5% formamide | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 1.5 | 24 or 23.7 | 30 |
3 | SplintR + 7.5% formamide | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 2.25 | 23.25 or 22.95 | 30 |
4 | SplintR + 10% formamide | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 3 | 22.5 or 22.2 | 30 |
5 | SplintR + 12.5% formamide | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 3.75 | 21.75 or 21.45 | 30 |
6 | SplintR + 15% formamide | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 4.5 | 21 or 20.7 | 30 |
7 | SplintR + 10% formamide + ET SSB | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 3 | 22.5 or 22.2 | 30 |
8 | Ampligase | 1.5 or 1.8 (0.3ul of each 10nM oligo) | 3 | 0 | 25.5 or 25.2 | 30 |
- Combine padlock probes and template in 1X Ligase buffer and possibly formamide
- Add mineral oil on top
- Incubate at 55C for 17hr