Matt:LabNotes/2017-4-7: Difference between revisions
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>Mzcai (Created page with "=SplintR RNA template specificity= ==Purpose== *Test to see if SplintR can ligate padlock probes in the absence of RNA templates *Concerned because [[Matt:LabNotes/2017-3-21|D...") |
>Mzcai m (→Protocol) |
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#Exo I/III digest | #Split each sample into 2 15ul samples | ||
#Purify all 10 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 both | #qPCR both | ||
#Run gel on both | #Run gel on both |
Revision as of 00:17, 9 April 2017
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 |
- 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 both
- Run gel on both