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===Mg++ protocol=== 1. Retrieve IVT RNA. *samples 1. Antarctic, SAP and PNK treated 2. Antarctic, SAP and PNK treated (meant to only do antarctic but forgot) 3. NTC 4. clontech control RNA used 2 uL each for samples 1, 2. Used RNA from #10 (top2) 2. Perform EtOH cleanup of RNA. (or do columns?) 3. Perform magnesium fragmentation (NEB) a. set up fragmentation buffer 1-9 uL Purified RNA 1 uL RNA fragmentation buffer (10X) X Nuclease free H2O b. Incubate in a preheated thermal cycler for 1-5 minutes at 94C. c. Transfer tube to ice d. Add 1 uL 10X RNA fragmentation stop solution 4. EtOH cleanup or RNA column purification *resuspend in 8 uL 5. Perform End repair of 3' End. (SAP, PNK, Antarctic Phosphotase) a. Add antarctic phosphotase 1 uL of 10X buffer 1 uL of 5U/ul antarctic phosphotase b. Incubate at 37C for 30 minutes c. mix SAP and PNK buffters together, before addition .5 uL of mixed buffer 1 uL SAP 1 uL PNK 2.5 uL nucleaes free H2O d. Incubate at 37C for 1 hour. (SAP needs 1 hour for 3' end phosphate removal) e. Incubate at 65C for 20 minutes for inactivation. 6. Poly(A) Addition with polyA polymerase (Enzymatics) *enzymatics PolyA polymerase. a. Add 2 uL of 100 uM DTT (with MMLV reagents) b. assemble reaction: 2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer) 1.5 uL polyA enzyme 1.5 uL 10 mM ATP (21 uL reaction now, in end will have ~50 mM Tris, 40 mM Kcl, 6 mM MgCl2, ~11 mM DTT, compatible with MMLV) c. Incubate at 37C for 10 minutes d. Heat inactivate at 70C for 20 minutes. (rui and NEB) 7. single strand synthesis MMLV RT (Clontech) *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 5 uL 20 uM primer stock to RNA sample. Final Volume = 25 uL (primer from BENG160 class, T20VN_PE_R) 2. Heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 2 uL 5X first strand buffer 3 uL dNTP mix 1 uL 100 uM DTT (keep at final [] at 10 uM) .75 uL SMART MMLV RT and mix (ADD LAST!!!!!) ____ 32 uL total 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes 8. second strand synthesis (qPCR) (KAPA) KAPA SYBR FAST qPCR mix X35 cycles 25 uL KAPA SYBR 4 uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24)) 1 uL H2O 20 uL DNA template (use 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 9.qiaquick cleanup OR beads purification (this time ran beads purification) *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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