Berglund:cold SHAPE method(Amy)

edited 5/25/10 AEM Cold RNA transcription using a SHAPE protocol (modified)

10x Transcription buffer 400mM Tris pH8.3 100mM MgCl2 20mM Spermidine 0.1% tritonX-100

Reaction mix: 158ul water 30ul 10X transcription buffer 60ul 25mM rNTP 6ul 400mM DTT 24ul 40% PEG8000 10ul PCR product (500ng) 12ul T7polymerase

300	final volume

Incubate in incubator for 1-1.5 hrs at 37°C Dilute to fv=600µl with water and add 6µl of 10x Rq1 Dnase buffer Add 3µl Rq1 Dnase (promega) and incubate for 1hr at 37°C

Clean using the Amersham/GE Q column protocol (http://openwetware.org/wiki/Berglund:RNA_column_purification) (changed protocol from dNTP to rNTP Oct 2009, whoops!)