Any ideas on the effective and economical method for total DNA extraction from intestinal contents in fish?
I perform a kind of RBB (repeat bead beating) method with silica columns :
Yu, Z., & Morrison, M. (2004). Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques, 36(5), 808–12. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15152600
Instead of Qiagen columns, I use columns from "EZ-10 Spin Column Animal DNA Mini-Preps Kit" which are far less expensive than the first ones.
Thank you Thomas! What kind of sample did you work with? Intestinal content or intestine tissue?
Hi,
I think this article may be able to help you, but you need to translate as it is in Chinese. Good luck!
Sorry, I can't attach the file. But the title of the article is 'Comparison of Three Methods for Total DNA Extraction of Intestinal Microorganisms in Puffer Fish' by LI Dan,PAN Ying-Jie,ZHAO Yong et al.FOOD SCIENCE, 2012, 33(9): 154-157.
Mahdi, I working on rainbow trout fry fed with various probiotics. I want to detect specifically these probiotics in the intestinal content but my samples are initially frozen (and small) so I try to detect the probiotics in the whole intestine tissue + intestinal content (= few milligrams of an ugly thawed stuff...). It works enough for my experiments but RBB extractions works far better with fresh stool and/or fresh intestine tissue.
There is other extraction methods, I advise you to look for Gatesoupe publications. He worked on fish total intestine microflora. He told me that DNA extraction can be different depending on development stage : extraction methods can be efficient on "old" fish but totally inefficient on younger fry (for the same species). Unfortunatelly, we must adapt extraction methods according the fish species, the organ, the development stage and of course the bacteria species.
You can see our latest publication in this book Dear Mahdi:
Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics
Hardcover: 416 pages
Publisher: Wiley-Blackwell; 1 edition (August 4, 2014)
Language: English
ISBN-10: 0470672714
ISBN-13: 978-0470672716
Good Luck
I found that commercial extraction kit - m.biotech is efficient enough to extract bacterial DNA from the gut samples. You may need to incubate your macerated samples with at least 20 ul lysozyme for overnight before performing the extraction protocol. Hope this help.
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