Plant DNA extraction - (Feb/18/2009 )
I work with plant molecular biology, and I'm having some problems to extract DNA of some particulary species. I've tried the CTAB protocol, I've tried the Ultra Clean Plant DNA Isolation Kit – Mobio, I've tried the Promega Kit, and some others protocols.
The species that I'm working with are from the genus Spondias, and here just few people works with that.
I'm desperate, I need to find an answer!
If somebody works with any specie of the genus, or if somebody have any answer for the problem, pleaseeeee contact me!!!
Send your mailing address to tech(at)aquaplasimd.com and I'll send you a trial sample of AquaGenomic. The protocol is here http://www.aquaplasmid.com/Protocol.html#plant. It should work for you.
you could try modification of the CTAB protoco
Look up DNA extraction protocols for Eucalyptus. Which is quite hard dues to a number of secondary metabolic products and carbohydrate. If the protocol can make head way with Eucalyptus, it probably can do some good for your plant. Here is an example
I'm not sure whether this genus has very thick cell walls, lots of lignin or does it produce many secondary metabolites ? Can you give us any more details about what makes this genus unique?
Do you get no DNA or just DNA that doesn't PCR well due to inhibitors?
I use the ctab protocol with modifications. In my specimens the problem is grinding the cells enough to break the cell walls, so I have to use special ground glass mortars, made from glass rods, in order to break open the cells.
Well, about the histochemistry characterization, we know that one specie have starch granule, oxalate of calcium crystals,
greasy composites, resins, phenolic composites and tannins. We also know that these species have citric acid and may have high polysaccharide levels.
I'm trying to extract DNA from 4 diferent species from the same genus. I can extract DNA from one especie (Spondias mombin), but the others I just can't. With the CTAB protocol, I did extract DNA once from 2 species (Spondias mombin, S. tuberosa), but the PCR failed. Actually, I didn't see the DNA on the spectrophotometer, only on agarose gel, so I didn't know exactly the quantity.
It looks like to me that despite they're all from the same genus, they're very diferent on their composition, wich probably causes the diference on dna extraction.
But that's the problem... I don't know how to do it!
I pm you a paper describing a way to produce PVPP spin columns (sorry no direct link to the journal); these are cheap and easy to produce in the lab. I use a bead beater and a CTAB method for extracting DNA from all kind of woody plants or timber.....and as a final step use these columns to purify the DNA; usually the results are good!
If you have the money: by a good quality taq specialised for difficult samples and try to include different amounts of BSA in your PCR usually this does the trick!