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Large-scale vitro transcription

Posted on Tuesday, October 31, 2006

Description
This will generate up to microgram quantities of RNA.

Procedure
-DNA (4ug plasmid or 200ng PCR fragment)
-20 ul 5X buffer
-1 ul 1 M DTT
-4 ul each NTP (100 mM stocks) (total of 16ul)
-2 ul 1 M MgCl2
-water to 100ul final volume
-5 ul T7 RNAP (~25 units/ul)

Incubate at 37 degrees C for 3-4 hours
37oC for 3-4 hours


Recipes
5X buffer
-0.2 M Tris-HCl (pH 7.5)
-50mM NaCl
-30 mM MgCl2
-10 mM spermidine


Supplies
RNase-free tips and tubes

Tips
Note that every needs to be RNase-free, so that your transcripts aren't immediately digested. See RNase-contamination prevention protocol.

It would be best to gel-purify the RNA after you make it, to get rid of unincorporated nucleotides and premature termination products.

Submitted by: hollings

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