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===protocol for and polyA tailing and RT-PCR (T7-top3)=== PolyA addition *enzymatics PolyA polymerase. 1. assemble reaction: 1 uL 10X polyA polymerase reaction buffer (or 2 uL 5X first strand buffer from SMART MMLV RT) 1 uL polyA enzyme 1 uL 10 mM ATP bring to 10 uL with RNA or w/e 2. Incubate at 37C for 10 minutes 3. Heat inactivate at 70C for 20 minutes. (rui and NEB) single strand synthesis *Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase] 20 uL reaction 1. Add 2.5 uL 20 uM primer stock to RNA sample. Brint to final volume of 11.5 uL with Nuclease free H2O (SMART CDS Primer II A this time) 2. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 4 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 uM DTT .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes second strand synthesis (qPCR) KAPA SYBR FAST qPCR mix X35 cycles 25 uL KAPA SYBR 4 uL primers, 1 uL F, 1 uL R (T7-top3-sss) and (PCR Reverse, Rui has) 1 uL H2O 20 uL DNA template (used whole RT reaction KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever *terminate when curves saturate qiaquick cleanup *run on gel or w/e *run qiaquick to clean sample before performing qPCR. **6 enzymes from 6 different reactions in there already.
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