[ashes11]

It seems that the DTT in NEB Buffer 4 precipitates COCl2 in the reaction solution. T-tailing of an internal control (50 ng PCR fragment, 360 bp) does not work in my hands when using NEB Buffer 4, 5mM COCl2 and NEB TdT enzyme. Here more proof from Schreiber Lab:

http://www.broadinstitute.org/chembio/lab_schreiber/pubs/protocols/IVT_Supplement/FAQ.html

[Kathi_seq]

Anyone actively using LinDA?

Hey,

I am struggling with the LinDA-protocol. Ashes11, did you find an alternative for the NEB4 buffer?
I have seen the brown precipitate after in vitro transcription.

I know these posts are pretty old, but maybe I am lucky and someone is still active here?

Thanks for your help in advance!

[Daytwa]

Sorry! It never worked for us.

[Kathi_seq]

Thanks Daytwa for your swift reply.

[Maflores11]

Hi Kathi_seq,

This is Miguel Flores (user: ashes11), I was unable to login in with my previous account.

In order to minimize COCl2 precipitation (likely due to DTT in NEB4 Buffer), I tried less concentration of COCl2 until I found a balance. I recommend to run an experiment with titrations of COCl2. I should probably contact my former adviser to check my notes to get specifics. LinDA worked in my hands, but requires some tweaking.

Cheers!