Berglund:cold SHAPE method(Amy)
From OpenWetWare
Jump to navigationJump to search
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!)
