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==Results & Conclusion== *Pos Ctrl (V6 probes capture gDNA with Ampligase) has low amount of ligated padlock probes **High Ct in PCR curve **I guess 300ng gDNA has fewer targets than 30ng RNA **But same low level as Neg Ctrl (Ampligase used on DNA/RNA hybrid) *NTC (V6 captures '''nothing''' with SplintR) has same amount of ligated padlock probes as Experimental samples (V6 captures RNA with SplintR) **suggests SplintR doesn't need RNA splint to ligate DNA *CONCLUSION: Need to check literature and/or call NEB but it seems SplintR has high ssDNA ligase activity, making it unusable for high specificity padlock probe applications **Checked and there is no reported ssDNA ligase activity, therefore it must be probes annealing to other probes **"Template-independent ligation" ===After meeting with Dr. Zhang=== *Reason PosCtrl does not show higher ligation is because forgot 95C is needed to denature gDNA! *He pointed out there is separation between NTC/30ng and 150ng/820ng PCR curves **Therefore there is some probe/splint ligation going on and conditions (eg temperature/time/concentration) need to be tuned to increase this separation ===Next Experiment=== *Find shortest amount of time necessary for SplintR Ligase at 37C **Minimize this time so less non-specific annealing of probes *Keep constant probe:target ratio for all *Try 3 NTCs with padlock probe concentrations matching 3 samples *Annealing temp: 95C 30sec -> Cool to 55C *Try different annealing times?
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