Matt:LabNotes/2017-4-7
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SplintR RNA template specificity
Purpose
- Test to see if SplintR can ligate padlock probes in the absence of RNA templates
- Concerned because DARTFISH in pure hydrogel (no RNA) resulted in some rolonies
- Also seems like only ppCUX2 rolonies
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 |
- Exo I/III digest 15ul from each reaction
- Purify all 10 samples
- qPCR both
- Run gel on both