Hello,
I am preparing RADseq libraries. I am shearing digested DNA (mean size 4096bp) using a Covaris ME220. The shearing is not working as fast as it should (compared to undigested DNA). Does anyone have any experience shearing digested DNA?
There are also the residuals from the digesting and ligations steps in the tube (rATP, enzymes, buffer etc). I assume this will also affect the efficacy, but I don't know how much. Hopefully I do not have to purify before shearing.
Thank you in advance for any help,
Ian
Hi Ian
you're working out the protocol recommended by covers, since the DNA size recommended must be higher than 10kB. you need then to use non digested DNA or to set up new time dose response to reach better treatments.
fred
Hi Ian,
As said up there, you need to choose a digestion enzyme that gives you longer fragments, as the success of shearing process decreases strongly when digested fragments are small. You can have a look at Davey et al. paper of 2013 (doi:10.1111 /mec.12084), they give a great explanation of every potential bias of RADseq and their consequences :).
Good luck with your RAD procedure !
What enzyme are you using for digestion, and how big is your genome? If digestion is really producing tiny fragments, it seems likely that you will end up with too many RAD loci to be tractable. Alternatively, you may have a problem with DNA quality. High quality starting DNA (i.e: the vast majority of the starting DNA is above 10KB) is essential.
Hello,
Thank you all for your suggestions!
I think we have fixed the problem (after talking with Covaris as well). Basically we had to run the Covaris machine for a lot longer than expected, but we are getting a pretty good spread of sizes around the required 500bp.
I should have added some more information. I am using a protocol that has been succesfully used before. However, the old protocol had a Biorupter in it, and our department has a new Covaris ME220 that we have been instructed to use instead (it is in the same building so a lot more convenient). Therefore I have been trouble shooting for that. In the previous protocol they were afraid of loss of DNA so did not purify (and it still worked). If i have any more problems, purification is going to be my first step.
Thank you again for the help
Ian
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