After ONE-STEP library PCR, column purification or ethanol precipitation?

We recently collected genomic DNA from a screen. We did PCR using about 3-5ug of DNA per well on a 96-well plate and got 5ml of PCR product to purify. So what to do next to purify the 300bp PCR band for Hi-seq? __ __

Do you do PCR purification followed by gel separation? I was wondering whether the large amount of genomic DNA (300-500ug) in the PCR mixture will overload the Qiagen columns, which has a binding capacity of 10ug. Or do you use ethanol/sodium acetate to precipitate both genomic DNA and PCR products, and then do gel separation?

Any advise would be much appreciated! __ __


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Lemon Cerberusalmost 4 years ago

I performe the first PCR step typically with 2.5 ug gDNA/well, then collect the crude PCR product and clean with Qiagen PCR purification kit (for cleaning 6 ml PCR crude (36 ml after adding PB buffer)) I use 5 Qiagen colums. Only then I subject the clean PCR product to gel electrophoresis separation and extraction to get a ready to go for sequencing purified PCR product.

Hope this helps,

Good luck,

Lemon Wyrmalmost 4 years ago

Thanks! We will try it.

Lemon Cerberusalmost 4 years ago

let me make a correction: It is 15 Qiagen columns needed for cleaning 6 ml PCR crude (36 ml after adding PB buffer), rather than the 5 that I have mistakenly typed before.

Sorry about the typo,

Khaki Cerberusalmost 4 years ago

From their 2017 paper: "For large-scale PCR purification, we recommend using the Zymo-Spin V with Reservoir."

Personally, I'm having trouble with the gel purification step. My 260/230 ratios are consistently ~0.1 by nanodrop and I don't know what to do. Qiagen, NEB, and Zymo kits all had the same result.

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