Making Long dsRNA from In Vitro Transcriptions


Posted by ychoi
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Protocol

Input DNAs:
Include phage promoter in the 5’-ends of your PCR products
-orlinearize
phage polymerase prooter vector (such as pBluescript).
Make the ssRNA:
Use mMessage mMachine kit [Ambion] to in vitro transcribe your DNA
Purify the RNAs on a denaturing polyacrylamide gel, UV shadow, and elute in model buffer (see
below). Precipitate and dissolve in H2O.
Make the dsRNA
ssRNA and asRNA [0.5microM] in 10 mM Tris HCl [pH 7.5] with 20 mM NaCl heated at 95C
for 1 minute
anneal at room temp for 12-16 hours
analyze between 100-500 ng on a 2% agarose gel TBE buffer (stained with Ethidium Bromide