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Old 11-16-2016, 05:14 AM   #1
IanAWarren
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Location: Cambridge

Join Date: Feb 2013
Posts: 11
Default Shearing digested DNA for RADseq

Hello,

I am making libraries for RADseq. I am trying out the Covaris ME220 for shearing the digested DNA. The shearing seems to be highly inefficient compared to what is given in the protocol book (using whole genomic DNA). Does anyone have any advice about ths in general?

I have identified three possible problems:

1) I am starting with digested DNA and I read somewhere that it does not shear as efficiently. The average size of my DNA should be 4096bp and I need to get it to ~500bp

2) Since I am going from the digestion to the ligation and then the shearing directly I still have the old enzymes, buffer, and some rATPs in the mixture.

3) I might be working with a low concentration of DNA, but I do not think this is right. I have about 1ug in 130ul, which is not low concentration to me.

I am going to perform some trial and error and hopefully find a solution, but my initial efforts have proved ineffective. I think i was being too tentative.

Thank you in advance for any help that you can provide
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