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Forum: RNA Sequencing 08-31-2017, 11:00 AM
Replies: 6
Views: 1,314
Posted By plyguy69
Hmmmm. I thought all MMLV enzymes had the...

Hmmmm. I thought all MMLV enzymes had the template switching activity. Anyway, in our hands the Primescript RT from Clontech gave the best results. As to your questions it seems like we get longer...
Forum: RNA Sequencing 08-31-2017, 05:59 AM
Replies: 6
Views: 1,314
Posted By plyguy69
Hi Radek. Thanks for the post. I am doing a...

Hi Radek. Thanks for the post. I am doing a very similar protocol currently and was wondering what RT enzyme are you using? Is it Superscript II/III?
Forum: Illumina/Solexa 03-23-2017, 05:27 AM
Replies: 29
Views: 7,341
Posted By plyguy69
Thumbs up thanks for the update

I've have also heard that the problem wasn't resolved, but never had details as to the cause.

Thanks!
Forum: Sample Prep / Library Generation 02-15-2017, 11:16 AM
Replies: 4
Views: 2,346
Posted By plyguy69
I've used both. And ClonTech's RTs. I've...

I've used both. And ClonTech's RTs. I've personally found that Maxima and Primescript both work well at 50C and 42C respectively. Try the Primescript with Betaine and DTT in the mix and see if...
Forum: Illumina/Solexa 01-31-2017, 07:31 AM
Replies: 29
Views: 7,341
Posted By plyguy69
Can anyone confirm this? I need to do some 2x300...

Can anyone confirm this? I need to do some 2x300 sequencing for Ig RNA-seq experiments. Would be awesome if I could use the MiSeq.
Thanks,
Forum: Sample Prep / Library Generation 10-17-2016, 12:34 PM
Replies: 6
Views: 2,816
Posted By plyguy69
We are still troubleshooting too

Hey guys,

We are trying to switch out our RT enzyme. Just tried the Maxima evolved RT from Thermo and the results for cDNA look promising. Need to carry these libraries through sequencing though...
Forum: RNA Sequencing 09-26-2016, 07:18 AM
Replies: 3
Views: 1,229
Posted By plyguy69
Thanks Nucacidhunter

Those cDNA products shown were analyzed after one SPRI bead clean up. You're right I can remove the majority of the lower bp nonspecific products by a second bead clean up (have tried and it...
Forum: RNA Sequencing 09-23-2016, 07:00 AM
Replies: 3
Views: 1,229
Posted By plyguy69
Unhappy Primescript Reverse Transcriptase Not Working

We are working on single-cell RNA seq using a version of Simone Picelli modified SMARTer method. Instead of Superscript II we were using Clontech's Primerscript. Unfortunately, the latest batches/...
Forum: Sample Prep / Library Generation 09-07-2016, 06:21 AM
Replies: 6
Views: 2,816
Posted By plyguy69
try changing RT enzyme

We are currently going through something similar but the nonspecific peak is shorter. We are trying to compare different RT enzymes to fix the problem.
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