# T7 HiScribe Kit RNA Synthesis

`PROTOCOL DUMP PLEASE FORMAT`

We can synthesize single-stranded RNA, from tens to thousands of nucleotides, by transcribing\_in vitro\_from a DNA template using T7 RNA polymerase. See[the NEB page](https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit)for more information.

**Constructing Your DNA Template**

[T7 RNA polymerase](https://en.wikipedia.org/wiki/T7_RNA_polymerase)is 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](http://salislab.pbworks.com/w/page/116137977/Anneal%20Single%20Stranded%20Oligos)(containing the T7 promoter sequence)

b)[PCR amplification](http://salislab.pbworks.com/w/page/115552054/Polymerase%20Chain%20Reaction%20\(PCR\))from 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

**T7*****in vitro*****RNA 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. Perform[phenol:chloroform extraction](http://salislab.pbworks.com/w/page/116201205/Phenol%3Achloroform%20RNA%20Extraction)or use[spin columns](http://salislab.pbworks.com/w/page/116201197/T7%20RNAP%20Reaction%20Cleanup%20Protocol)to remove excess nucleotides, enzyme, and salts from the reaction.