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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:
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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!Comment
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Sorry! It never worked for us.Comment
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Thanks Daytwa for your swift reply.Comment
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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!Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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