By P.C. van der Vliet (Eds.)
The relevant 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 booklet presents scientists with a accomplished number of completely verified updated manuals for investigating RNA-protein complexes in vitro. The protocols may be played through researchers knowledgeable in usual molecular organic ideas and require at the least really expert apparatus. The systems contain suggestion of providers of reagents.
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Extra info for Analysis of RNA-Protein Complexes 'in vitro'
5. b 6. Place 2 ml CsCl cushion at the bottom of a polyallomer ultracentrifuge tube (14 x 89 mm). 7. Layer the homogenate carefully on top of the cushion. 8. ' 9. Remove the majority of the supernatant with a 10 ml disposable pipette taking care not to touch the bottom of the tube. Pour off the remainder of the supernatant and leave the tube in an inverted position. 10. Cut off the bottom of the tube with a heated scalpel. 11. 1 autodigesteddproteinase K solution and transfer to a microfuge tube.
1. 5 M Na2HP04 solution (nontitrated) Procedure 1. 1) with 18 pl H20. 2. Spot 8 p1 to each of two Whatman DE-81 filter discs. Air dry. 3. Place one filter in a scintillation vial and count (count A). 4. Swirl gently for 2-3 min. Pour off buffer. 5. Repeat wash 2-3 times. 6. Wash twice in H 2 0 for 30 s each time. 7. Rinse in EtOH, dry and count (count B). Assuming equal representation of the different nucleotides. 24 x (count B/count A) x (nmols cold UTP in the reaction). 2. Cotranscriptional labelling or modiJication of RNA Radioactive or modified nucleotides can be incorporated randomly in a transcript or specifically at the 5’-position of the transcript during transcription.
A critical step in the procedure is the redissolution of the RNA pellet after ultracentrifugation, since the denaturants are absent at this stage. Therefore, the inclusion of an autodigested proteinase K solution provides a ribonuclease-free environment during this step and has the added advantage of degrading any proteinaceous material in the pellet. The glass-clear RNA pellet at the bottom of the ultracentrifugation tube is sometimes difficult to see, but after the proteinase K solution has been added the release of the pellet can be observed.