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Example of double digest gel photo or BioAnalyzer trace

Posted by marceltinning on 23 Apr 2014 at 10:18 GMT

I wondering if authors could be asked to submit some information and gel photos in supplementary materials regarding expected results after each or important steps in their library preparation method. That will help others to troubleshoot their prep or compare results with published methods. For instance, a BioAnalyzer trace or gel photo after single or double digest and final library would be very helpful for many researchers who are trying to adopt this paper's method resulting in increased citation for the paper. I am sorry to see that in this paper the authors even have not mentioned the concentration of their adapters and PCR condition that they used for the presented results. I appreciate that they have provided many online tools for proper implementation of the method. Their online protocols also lacks any examples. I hope that editor could post extra information in this regard as a comment or make it a policy for future publications that present a new method.

Kind regards,

Marcel Tinning.

No competing interests declared.
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RE: Example of double digest gel photo or BioAnalyzer trace

agottscho replied to marceltinning on 29 Oct 2014 at 18:27 GMT

Hi Marcel,

I am not an author on this paper but I have successfully used this method for several manuscripts currently in prep. Regarding your questions, first for the gel photo of the double digest: I simply run out a 1% gel with digested and undigested DNA to compare them. The digested DNA should appear more "smeary" and although I still see a high molecular weight band remaining sometimes, which worried me at first, I nevertheless always obtained enough digested fragments to get lots of good data back in the end. Second, the concentration of adapters depends directly on the number of fragments (loci) expected, which is in turn based on the size of the genome and the enzymes you used - the authors provided a ligation molarity calculator in the supplementary material to address this. Finally, regarding the PCR conditions, the standard thermocycler protocol is listed in the Phusion PCR kit. You don't really know that anything worked until after the PCR step - you should be able to run it out on a gel and see a band at the size range you selected. A Bioanalyzer run should confirm that. Hope that helps.

Best,

Andrew Gottscho
PhD Candidate, Evolutionary Biology
Department of Biology
San Diego State University
5500 Campanile Dr
San Diego, CA 92182

No competing interests declared.
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