Thanks both and yes, absolutely agree. We use NEB Stable E.coli to reduce the loss of repetitive sequences, but we seem to have lost ~90nt. PCR is not an option for us at this point due to amount of product we would need. We are looking at RCA in the future. - ThreadSky

cmordstein.bsky.social • 103 days ago

Thanks both and yes, absolutely agree. We use NEB Stable E.coli to reduce the loss of repetitive sequences, but we seem to have lost ~90nt. PCR is not an option for us at this point due to amount of product we would need. We are looking at RCA in the future.

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jamespblloyd.bsky.social•103 days ago

We have used NEBstable a lot but not seen any difference to other cells (NEB 10beta) in terms of stability. Sadly we have not characterised it in depth, and the issue is for repeated units rather than long A stretches. But we found growth at 30C rather than 37C helped (NEBstable did not help!)

cmordstein.bsky.social•103 days ago

Thanks James. Do you have a recommendation for a particular strain or is the conclusion that they are are all equally good/bad?

jamespblloyd.bsky.social•103 days ago

From our anecdotal evidence, all equally bad. But growing at colder temp seemed to help us. But I have never done anything with DNA encoded polyA. But I’d be curious about the outcome.

cmordstein.bsky.social•103 days ago

Thanks a lot, we will test the lower tempt for sure. At this point, I am hoping this is a nanopore artefact. The CRO we ordered the plasmid from should have QC'd the length of the tail as we paid for that... We are currently waiting to receive their report.

sethcheetham.bsky.social•103 days ago

How much mRNA are you producing? We routinely use PCR to generate templates for up to 1-20mg productions. I know people that have done larger batches too.

cmordstein.bsky.social•103 days ago

For routine production, we are usually more in the 30-50mg range. I should say we do IVT in flow, not batch. How are you dealing with PCR errors at this scale?

sethcheetham.bsky.social•103 days ago

We use q5 which has a very low error rate. We've done a lot of ONTseq of plasmid and pcr templates and don't see any increase in error rate, but we see way better polyA integrity.

cmordstein.bsky.social•103 days ago

Interesting and thanks for sharing! I will have a think.

sethcheetham.bsky.social•103 days ago

Happy to sure our protocol if helpful, just send me a DM

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