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Hello, I have a basic question. I am extracting samples that will be around 3.5 Mb and only done sonication on whole genome. Can I still use it with the smaller input or will I over fragment? Is enzymatic a better way to go?
Thanks
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Are you using Covaris? I think it wouldn't be a problem because you can regulate many things with the settings of the machine. I myself sonicated fragments around 100 to 10 kb end ended in the library range (300bp) with the settings given by Covaris. -
Large DNAs will fragment during isolation, so after isolation your 3.5 Mb DNA will most likely have the same fragment size distribution as you would get from purifying larger genomes. There should be no difference in further fragmentation (Covaris, other sonication, etc.) between the two. So, short answer, you shouldn't need to change any conditions for fragmenting.
Where things get tricky is on the low end. In our hands, Covaris works pretty well with fragments as small as ~1 kb, but smaller than that and you start to run into problems. 400 bp amplicons really don't fragment at all in the Covaris. I'm sure there is a good biophysical explanation for this, but we have just seen this empirically.Comment
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Yes, planning on using Covaris. Thanks so much for your help!Comment
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You may also want to try Fragmentase from NEBComment
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