T7 HiScribe Kit RNA Synthesis

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We can synthesize single-stranded RNA, from tens to thousands of nucleotides, by transcribing_in vitro_from a DNA template using T7 RNA polymerase. Seethe NEB pagefor more information.

Constructing Your DNA Template

T7 RNA polymeraseis a highly processive RNA polymerase from T7 bacteriophage. T7 requires a specific promoter sequence (which is significantly different from E. coli promoters).

T7 Promoter:

TAATACGACTCACTATA|GG

            +1^note that T7 requires at least one G \(preferably 2\) at the very 5' end of the transcript

No modification is required for the rest of your sequence. No terminator is required; T7 will fall off the end of the DNA template.

The template can be constructed by:

a)Annealing 2 DNA oligos(containing the T7 promoter sequence)

b)PCR amplificationfrom an existing template. This is a convenient way to add the T7 promoter to a sequence which does not currently contain it, by adding the promoter sequence to the 5' tail of your forward PCR primer

Cleaning Up Your DNA Template

The DNA template that goes into a T7 reaction should be free of RNases, to ensure high yield of RNA product. Take the following precautions when handling your DNA:

If you_anneal_two DNA oligos:

• Resuspend with DEPC water (which is RNase-free)

• Use RNase-free filter tips, and work at the RNA bench

If you_PCR amplify_a DNA template

• Once the PCR is complete, use RNase-clean technique to perform a PCR cleanup

T7in vitroRNA Synthesis

Note: use RNase-clean technique

Combine the following:

DNA Template	500 ng
NTPs + Buffer	10 uL
T7 RNA Polymerase Mix	2 uL
DTT (1 M)	0.3 uL
ddH2O	to 30 uL

Flick to mix, and spin down. Incubate for 16 hours at 37 C. Performphenol:chloroform extractionor usespin columnsto remove excess nucleotides, enzyme, and salts from the reaction.