We are trying to fragment genomic DNA using NEB's Fragmentase enzyme with a 20-23 minute incubation. Our goal is to reach fragments near 300bp for sequencing on GAIIx. Has anyone used this enzyme and if so what are your thoughts on it? When cleaning with a Qiagen Qiaquick kit, I'm getting recoveries of 35-40% initial starting material (starting with 1-5 ug). I'm thinking that a lof of DNA may be chopped small and then not caught by the columns, they don't capture fragments under 70bp, but I am not able to confirm this. Thanks!
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We have not tried Fragmentase yet.
Your estimation of the amount of starting DNA is based upon what assay? Unless you use fluorimetry or visualization (on a gel) against a known mass standard, then it is likely your will over-estimate the amount of starting DNA you have. There are lots of confounding contaminants in a typical DNA prep (RNA -- whether you RNAsed or not, and trace phenol being the two I most commonly see.) UV spectrophotometry will almost always give you an over-estimate for this reason. (After fragmenting your purification step will largely remove these contaminants. Hence the low apparent yields.)
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Phillip -
Oops, I forgot to include that. We quantified our DNA via Qubit Flourometer prior to fragmenting AND after.Comment
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Then you are likely correct. Although it is possible the Qiaquick columns have lower capacity than you expect.
You could run aliquots from a sample before and after Qiaquick on a gel or bioanalyzer chip to verify.
Mechanical fragmentation methods become more inefficient at shearing as they approach their lower size limit. As a result it is possible to tune them to put a large percentage of fragments above a size limit. With an enzyme there would be no real physical limitation on cleaving smaller and smaller fragments. So losing a higher percentage of your DNA on the low end may part of the price you pay for using an enzymatic method.
On the plus side, the fragment ends may be more ligate-able after enzymatic fragmentation. Possibly.
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PhillipComment
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How much elution buffer are you using to elute your sample off of the Qiaquicks? There is a pretty direct correlation between the amount used to elute and the elution recovery. See the attached file.
Hope that helps,
JenComment
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I was eluting with two washes of 30 uL in a QiaQuick column and diluting to a final volume of 100 uL. I read similar information about the columns as what you posted and have changed my protocol to one wash with 100 uL. The protocol you posted is much more detailed and useful than the one I found. Thanks a bunch!Comment
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No problem, glad to help.Comment
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We tried using this enzyme, we had it automated on a Biomek Fx and it seemed to work OK the first time we did the expt. Then we repeated everything using some 48 samples and everything failed, I contacted NEB and they first tried to tell me I hadn't mixed the enzyme properly so I told them there was a mix step incorporated into the robot program. In the end they gave us our money back and we now use a Covaris, it seems the enzyme is very unstable and doesn't give consistent results. My advice would be to try something different if you can.Comment
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I have used NEBnext fragmentase for approx 300 bacterial genomes. Works well!Comment
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