By P.C. van der Vliet (Eds.)
The primary function of RNA in lots of mobile methods, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This ebook presents scientists with a complete selection of completely proven up to date manuals for investigating RNA-protein complexes in vitro. The protocols might be played by way of researchers expert in common molecular organic strategies and require not less than really good apparatus. The approaches comprise suggestion of providers of reagents.
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Notes a. 1% antifoam A. b. Do not use neutralised phenol. c. The volume is unlikely to exceed 1ml. d. The ethanol precipitation step is introduced to remove traces of guanidinium thiocyanate. Comments Since the guanidinium thiocyanate acid-phenol method can process Ch. 2 PREPARATION OF R N A 25 a large number of samples on a small scale, several manufacturers base their total RNA isolation kits on this method. Therefore, efforts have been directed towards simplifying the original procedure. The major short-cut is to incorporate phenol in the lysis solution, but the kit protocols tend to stick with the original ratio of 1ml lysis solution per 100 mg tissue or lo7 cells.
1 H20. Ch. 2 PREPARATION OF RNA 43 4. Add 1 ~ 51mg/ml tRNA ( E . coli RNase free) or glycogen ( 5 mg/ml) if tRNA is critical. 5 . ‘ 6. Extract l x with chloroform. 7 . d 8. Redissolve in H20. Notes a. Add 5 m M MgC12 (in addition to MnC12) for incorporation of 2’-modified CTP. b. The fraction of modified NTP in the transcription mixture to obtain 5% incorporation should be approximately 50% for dCTP and dTTP, while more than 75% of the nucleotide should be modified for dATP, dGTP, [as]dNTP or 2’-O-methyl NTP.
The yield of extracted RNA can be improved by including 2 g acid-washed glass beads. c. A combined heating block and shaker such as the Eppendorf 5436 thermomixer is useful. Ch. 2 PREPARATION OF RNA 27 d. 4) followed by an additional ethanol precipitation. However, the LiCl precipitation will jeopardise the yield of RNAs <200 nucleotides. 4. 5 10 mM MgClz 1 mM EDTA 10 units DNase I (RNase-free from Pharmacia). 3% SDS. Equipment Swinging bucket microfuge Procedure 1. Rinse the dish (100mm) twice with 10ml ice-cold PBS.
Analysis of RNA-Protein Complexes 'in vitro' by P.C. van der Vliet (Eds.)