T7 RNA Polymerase

What it means: Concentration is how much enzyme activity is packed per microlitre. At 50 U/µl, one unit incorporates 1 nmol of ATP into acid-insoluble material in a 50 µl reaction over 1 hour at 37°C.

Why it matters:

• Dosing by activity rather than by volume is what keeps transcription yields reproducible between lots.
• At 50 U/µl the enzyme is concentrated, so typical reactions need well under a microlitre — pipetting error becomes the dominant variable.
• Adding more enzyme than needed increases the glycerol carried into the reaction, which can itself suppress transcription.

How to interpret here: The 100 µl pack supplies 5,000 U in total, covering a large number of standard reactions. Scale enzyme to template amount, not to reaction volume.

Tip: For sub-microlitre additions, make a working dilution in cold 1X buffer rather than pipetting fractions of a microlitre.

What it means: Promoter Specificity describes which DNA sequence the polymerase will start from. This enzyme initiates only at the T7 phage promoter and does not recognise SP6 or T3 promoter sequences.

Why it matters:

• Strict specificity means transcription starts only where you placed the promoter — no background transcription from elsewhere in the template.
• It allows one strand of a construct to be transcribed selectively when different phage promoters flank an insert.
• A template without a correctly oriented T7 promoter immediately upstream of the target will produce nothing at all.

How to interpret here: Confirm the T7 promoter sits immediately upstream of the region to be transcribed and in the right orientation. If a construct carries SP6 or T3 sites, this enzyme will ignore them.

Tip: Linearise a plasmid template downstream of the insert; on a circular template the polymerase runs on and produces heterogeneous long transcripts.

What it means: Buffer Included describes the reaction buffer supplied with the enzyme — 1.0 ml of 10X T7 RNA Polymerase Reaction Buffer, matched to the 5,000 U enzyme pack.

Why it matters:

• In vitro transcription is sensitive to salt, pH and magnesium; a generic polymerase buffer will not reproduce the yield the enzyme was characterised at.
• The matched buffer removes composition as a variable when a transcription reaction gives low yield.
• Buffer volume is scaled to the enzyme pack, so nothing runs out mid-project.

How to interpret here: Dilute the 10X buffer ten-fold in the final reaction. NTPs are not supplied and must be added separately.

Tip: Assemble transcription reactions at room temperature rather than on ice — spermidine in transcription buffers can precipitate the template DNA when cold.