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==Results== ===Before Exo I/III Digest of Unligated Padlock Probes=== [[File:20160909_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160909_SplintRTest2_qPCR_Digested.JPG|350px]] ====Template Digested Notes==== *Biggest factor for ligated products is concentration of padlock probes added **Suggests there is a lot of non-specific annealing between padlock probes at higher concentrations *Separation between NTC and RNA template samples increases as padlock probe concentration decreases **30ng samples looked best, with NTC less than NegCtrl *PosCtrl less ligated products than NegCtrl is an anomaly **Probably cause: NegCtrl had 2.5X more padlock probes added than PosCtrl **15min of Ampligase is not enough time for PosCtrl to have a lot of ligation compared to normal protocol of 24hrs ====Template Not Digested Notes==== *Theoretically should be the same? Since RNA template cannot be amplified by KAPA polymerase and thermocycling should denature padlock probes from template **Maybe PCR primers are occupied with template *Separation between sample and NTC for 820ng and 30ng almost non-existent but 150ng still has good separation **I could see more template leads to PCR primers being occupied on template, which is why 820ng sample amplifies less than NTC but I would then expect 150ng to show more similarity to that than 30ng *PosCtrl and NegCtrl look "correct" ===After Exo I/III Digest of Unligated Padlock Probes=== [[File:20160912_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160912_SplintRTest2_qPCR_Digested.JPG|350px]] ====Template Digested Notes==== *This is the "correct" experiment **Everything points to using less padlock probes limits false positives in NTC *SplintR is much more active than Ampligase ====Template Not Digested Notes==== *Oddly this shows the ~30nM Padlock Probe concentration of 150ng samples is the sweet spot **No idea why there is such a large separation if RNA is not digested
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