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Has anyone used AMPureXP for ssDNA purification? I need to get rid of smaller cDNAs (<100 ntd).
Biochembug
Has anyone used AMPureXP for ssDNA purification? I need to get rid of smaller cDNAs (<100 ntd).
Biochembug
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Yes it works for ssDNA. It also works for RNA. And DNA/RNA hybrids.--------------
Ethan -
What volume should I use to eliminate low molecular weight (<100 ntd) cDNA?Comment
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What I do is I mix 1 volume of DNA or RNA with 1 volume of AMPure XP and 1 volume of 30% PEG800 1.25M NaCl. That gives me a cut off of about 100 bp. If you search the forum there is a thread with more information on this with some guy that posted a nice experiment. Be warned that there is batch to batch variation with the AMPure XP beads.
There are some protocols on my blog at http://ethanomics.wordpress.com/ which perhaps describe what I do a little better.--------------
EthanComment
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Thanks for the info Ethan. Will give a shot!
BiochembugComment
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Hi Ethan,Originally posted by ETHANol View Post
Can you comment on what the cutoff seems to be for dsDNA vs ssDNA? Does it appear to be a function of molecular weight, strand length, or something else?
PhillipComment
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There's a recent post on James Hadfield's blog detailing how the beads work and how the concentration of PEG/NaCl effects the size selection.
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Hi all,
Great to see that you guys are keeping this thread alive. As pointed out by Phillip, I am looking for the cut-off of the single stranded DNA molecules on AMPure beads. Comments are always welcome!!!
Thanks TonyBrooks for that like.
BiochembugComment
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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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