AFLP requires a highly purified and high quality DNA. In the case of plants which are rich in polyphenols and polysaccharides, what are the best methods for DNA extraction?
It depends on the species in question, I am afraid. Standard CTAB methods can work well, QIAGEN kits, Machery-Nagel kits also work well with various plants. Try to get some free trial kits and then compare for quality and quantity of the resulting extracts. [Apologies if this posts twice ... my link seems to be playing up].
It depends on the species in question, I am afraid. Standard CTAB methods can work well, QIAGEN kits, Machery-Nagel kits also work well with various plants. Try to get some free trial kits and then compare for quality and quantity of the resulting extracts. [Apologies if this posts twice ... my link seems to be playing up].
Hi, I agree with Susan. For my AFLPs I extracted the DNA from the codling moth (Cydia pomonella) with a modified CTAB-method protocol. I didn't test different methods. Cheers, Nadine
Thanks all! I'm working on genomic DNA extraction from silica-dried leaves for a plant has a high content of phenolic compounds and polysaccharides. There is no problem with the young seedlings of this plant. For the trees, I found the following problems:
1. For the first extraction, I got a yellowish DNA due to the phenolic compounds even I'm using 2 % CTAB buffer plus urea, boric acid, and Mercaptoethanol. I was precipitating organic compounds with chloroform Isoamyl (24:1) 2 times.
2. For the second extraction, I tested phenol: chloroform Isoamyl (25: 24:1) and Ammonium acetate but I got a gelatinous pellets. AFLP reaction with this methods wasn't bad but I'm looking forward better results. Based on your great experiences, any other method can be used.
Thanks a lot for your interest!
In our lab, we use the DNeasy kit (Qiagen) for genomic DNA extraction, with some variations of the original protocol. We work testing AFLP in different forest species, so the DNA obtained by this method, has a high quality. I currently work with MSAP (a variation of AFLP), and I've had good results with the genomic DNA extracted by the Qiagen kit. Using this method, it's easier and faster than CTAB protocol.
To our experience, methods based on magnetic bead (like MagJET DNA Purification Kits) give the the most stable and independent of organism type result.
But, in skilled hands, classic CTAB-SDS method can be useful as well.
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