Rnasin

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube 1.0 pL RNasin (RNase inhibitor), 11.5 pL UV-irradiated dH20, 37.5 U DNase (RQl RNase free DNase, Promega Inc., Madison, Wl)/(1 U/pL) Total (per slide) 50.0 pL. 

Nuclease-free dd O T4 DNA ligase (Promega) 0.317 Weiss U pL RNasin (Promega) 1 U pL-1 Modified, purified transcript of RNA HPLC-purified 10-mer-PEG substrate 20-mer bridging oligonucleotide... 

Add this buffer/water/RNasin/T4 mix to the annealed ligation components and subject it to the following thermocycler sequence ... 

RNasin Plus RNase inhibitor, Promega (N2615)... 

RNase H buffer, 12.5 fil RNasin Plus RNase inhibitor, and 50 /(I RNase H enzyme to 137.5 fil nuclease-free H20. Add this to the annealing reaction. Incubate at 37 °C for 2 h. Stop the reaction with l/20th volume 0.5 M EDTA, pH 8.0. 

The mRNA is then precipitated with 2.5 volumes ethanol and dissolved in 10 pi H2O containing 10 mM dithiothreitol and 0.1 U/ pi RNasin... 

If positive results are seen after probing, repeat the digestion by DNase. Add 10 pg of RNA template to a solution containing 20 U of RNase-free DNase, 1.5 U of RNasin, 50 mM of MgCl2, 5 mM of DTT, and 2 mM of ethylene glycol-bis (3 -aminoethyl ether)-/V,/V,/V,/V-tetra-acetic acid (EGTA). Mix well and incubate for 60 min at 37°C. 

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