Hi All,
I'm thinking about to do GBS, but I am not quite sure how good my DNA samples should be, in terms of integrity. I think I have good amount of DNA, but some of my DNA samples may have degraded. Does anyone know anything about the DNA quality that is required to do GBS?
Thanks!
Jing
Which type of sample will you be working with?
If your DNA samples were extracted from whole blood or mouth swap you shouldnt have any problems in regard to the integrity of the samples.
I'm not very familiar with genotyping, but i have been working with Bisulfite-treated DNA samples (which is a quite harsh treatment that compromises DNA integrty) and i didn't have mayor problems when analyzing them by Sanger Sequencing, eventhough my DNA samples where first extracted from ffpe tissue samples, which are normally highly degraded.
Hi Mauricio,
Thanks for your answer!
I work with an oomycete, it is a microorganism but phylogenetically more close to plants. I did a tapestation analysis with some of my DNA samples, the result was not that good. Most of the samples have largest band around 15kb, which is smaller than the size of genomic DNA we usually see.
I guess what I am concerned about is that the DNA degradation will affect the enzyme digestion. I would guess degraded DNA sample has much less restriction sites.
Thanks,
Jing
Jing,
I think the integrity of DNA is very crucial. If I were you I would rather put more effort into redoing the extraction to get better DNA quality, to be safe. About the degradation of DNA, you could do a couple of things to check. You could check if there is any smearing of the DNA. If no smearing is seen and if it is a big band at the top of the gel, the DNA could be okay (make sure to run long enough so that the 15 kb band also moves some distance in the gel). Another thing you could do to confirm purity is to check the 260/280 ratio of the DNA.
Good luck,
Sirjan
Hi Sirjan,
Thanks a lot !
I sent some of my samples to do the tapestation analysis, most DNA samples have largest bands ranged from 7kb to 15kb, I can clearly see the smearing of DNA from the picture I got back. I was wondering what size of band would you expect for genomic DNA, around 20kb-30kb or even larger?
I am planning to re-extract the DNA. Do you have tips of extracting genomic DNA or do you have any recommendation of any genomic DNA extraction Kit?
Thanks again!
Jing
Jing,
Genomic DNA forms a large band, probably larger than any ladder size. You can tell by judging your highest band. If the band is intact, without smear and higher than you highest band in ladder, I think that should be good.
I think re-extracting is a good idea. I work with plants. We use Qiagen's DNeasy plant kit for DNA extraction. I don't know if it is good for fungal/oomycete samples. I am sure there are kit around to extract fungal DNA and you could find about that in MPMI or any publications with fungal experiments. It is good to use kit to get DNA for high throughput stuff like GBS where purity is of prime importance.
Good luck,
Sirjan
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