Matt:LabNotes/2017-4-7
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SplintR RNA template specificity[edit]
Purpose[edit]
- 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[edit]
- PP + gDNA + SplintR
- PP + SplintR
- PP -ppCUX2 + SplintR
- PP (negative control for qPCR)
- PP + CircLigase II (positive control)
Protocol[edit]
- 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
- Elute 15ul each
- 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
File:20170408 SplintRNoTemplateTest qPCR.PNG
- Run gel on both
- PP + gDNA + SplintR
- PP + SplintR
- PP -ppCUX2 + SplintR
- PP (negative control for qPCR)
- PP + CircLigase II (positive control)
- PP + gDNA + SplintR (digested)
- PP + SplintR (digested)
- PP -ppCUX2 + SplintR (digested)
- PP (negative control for qPCR) (digested)
- PP + CircLigase II (positive control) (digested)
File:2017-04-08 SplintR DNAsplint Test.jpg
Conclusion[edit]
- SplintR ligates padlock probes on DNA splints at slightly less efficiency than Circligase II
- Negative control worked: linear padlock probes can not be amplified with these PCR primers
- Exo I/III digestion improved efficiency of PCR of circular templates
- SPECIFICITY MUST BE CONTROLLED WITH HIGH TEMP ANNEALING AND THOROUGH WASHING