Hi. I've been using fin clips from zebrafish for genomic DNA extraction, followed by PCR, and then ExoI and TSAP treatment. I then send these off for sequencing. So far I've been using this method to determine the genotype of my fish for my gene of interest. Recently though I was advised by someone that this is, in fact, a "short cut" strategy.. Essentially, what they suggested is transforming the PCR product, picking 10 colonies and sending them all for sequencing. This gives a very reliable readout.
However, this would mean 10 samples per fish and I've multiple tanks to genotype, making it not only time consuming but 10 times more expensive.
How does one make the decision between the two methods? In my case, since I'm only wanting to genotype, is my method sufficient?
Dear Kunar
Your method is sufficient. Sequnecing cloes will not lead to more confident results. But have a look at the blast search. Here a possible publication to judge such searches. You can download the publication from my researchgate.
Success
René
Hartmut Rehbein, René Köppel and Thomas Hankeln (2012) Leitfaden für die Lebensmittelüberwachung zur Identifizierung der Fischart durch DNA-Sequenzierung von PCR-Produkten , Journal für Verbraucherschutz und Lebensmittelsicherheit Identifiers Vol 7 (3):255-260
Dear Kunar,
You have used the right method for genotyping. You may go head with PCR genotyping follwed by 10% samples for sequencing for validation purpose.
All the Best
Rajan
Thank you very much René, Rajan and Artur! Also, thank you for the publication references.
Dear Kunal,
It’ not exactly my field, but I just have an idea. If the gene of interest is between two linked markers (eg. SSRs), than I think that the most cost-efficient (but maybe “old-fashioned”) way is to use them. Sometimes older techniques are sufficient and much cheaper.
Regards, Gizella
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