Hello everyone,

This involves New England Biolab kits.

HiScribe® T7 ARCA mRNA Kit (without tailing) NEB #E2065S

https://www.neb.com/en-us/products/e2065-hiscribe-t7-arca-mrna-kit

HiScribe® T7 ARCA mRNA Kit (with tailing) NEB #E2060S

https://www.neb.com/en-us/products/e2060-hiscribe-t7-arca-mrna-kit-with-tailing

I'll refer to them as '65 (without tailing) and '60 (with tailing). I tried to number my questions as well.

Both kit manuals describe the ability to produce tailed mRNA, but they are not clear on what element has to be present in the DNA template to produce the Poly(A) tail.  The '65 (No Tailing) kit explicitly describes that tailing is included in the In Vitro Transcription (IVT) step using the T7 RNA Polymerase as long as the plasmid encodes a Poly(A) Tail. 

The '60 (with tailing) kit includes a separate reaction for the tailing step using E. coli Poly(A) polymerase, but uses the same T7 RNA Polymerase for the IVT.  1. Our main question is what exactly does it mean to "encode" a Poly(A) tail in this context? What is the T7 RNA polymerase recognizing in the DNA template to add a tail? A poly(A) signal or a series of Ts? Or something else entirely different? 2. Can the '65 (without tailing) automatically produce a poly(A) tail based on the DNA template having a Poly(A)-Signal-Sequence like AAUAAA? Or does encoding mean that the DNA template needs to literally encode a series of Ts to get a Poly(A) tail?  3. Is the '60 (with tailing) kit meant for DNA inserts that are missing a poly(a) signal or series of Ts? My Lab has three plasmid situations but this ambiguity is confusing us on which kit to use for the individual plasmids.

Plasmid A (or plasmid 'PAS')

• has the DNA insert encoding a AAUAAA signal near the end before the T7 termination signal.

Plasmid B (or plasmid 'PolyT')

• has a DNA insert containing a series of Ts that would produce a Poly(A) of predetermined length before the T7 termination signal. 

Plasmid C (or plasmid 'truncated')

• has a DNA insert containing NO poly(A) signal or series of Ts before the T7 termination signal.

4. For each of these plasmids above, which kit would be ideal?

Without knowing how '65 (without tailing) recognizes to add a tail, we don't know which kit to use for each of them.

Thanks everyone,

-A