Ribonuclease 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... 

Compared with DNA, more care has to be taken when working with RNA to prevent any RNase contamination. Use of gloves (hands are the major source of RNase), diethyl pyrocarbonate (DEPC)-treated solutions, disposable tubes, pipet tips from freshly opened bags, and RNasin (a potent ribonuclease inhibitor) in the labeling reaction, minimizes the risk of RNA degradation. 

RNasin ribonuclease inhibitor (Promega, Southampton, UK). Store at -20°C. Restriction enzymes (single cut 5 and 3 of insert). 

In Vitro Translation. TnT Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used according to the manufacturer s instructions. 25 rabbit reticulocyte lysate was added to 2 p of TNT Reaction Buffer, 1 of RNasin ribonuclease inhibitor (40 u///l), 1 of amino acid mixture minus methionine (1 mM), 2 /il [ S] methionine (>1000 Ci/mmol at 10 mCi/ml), 2 of DNA template(s) (0.5 pgJpV), 1 /.tl of T7 polymerase, and 16 /il of nuclease-free water to a final volume of 50 /il. The reaction was incubated at 30° for 1 h. 

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