Problems isolating plant genomic DNA
Posted 17 February 2005 - 11:18 AM
We are trying to extract genomic DNA from the leaves of an Ericacea (we suppose it is rich in tanins and phenolics) and we can't do it!
We have used two different kits which work OK for other plant species but fail with this one. With the kit from Nucleon we see a big pellet (DNA+contaminants?) and when we run that on a electrophoresis we can't see anything!!
Can anyone suugest anything?
Posted 20 February 2005 - 04:19 AM
i've never worked with plant DNA, but can you use the traditional phenol/chloroform DNA extraction method? we have had few cases of bacterial species were kits didn't work and this method did.
and a tip - Eppendorf makes tubes with "phase-lock gel" which are really helpful for extractions - check if they gave one compatible with plant DNA extraction.
Posted 20 February 2005 - 07:48 PM
I used to extract DNA and RNA from Pittosporaceae, which are very rich in phenolics, using a standard PCI (Phenol:choloroform: Isoamyl alcohol) extraction, which worked very well. However for this extraction we were typically using most of a leaf (maybe 500mg) I can't remember exactly.
The main problem was that often sugars etc co-precipitated with the DNA and resulted in a huge pellet that was hard to dissolve and often discolored. Repeat extractions usually solved this.
best of luck
Edited by bob1, 20 February 2005 - 07:49 PM.
Posted 21 February 2005 - 04:16 AM
We have tried now using the CTAB protocol and double phenol: chloroform extractions, we got a nicer cleaner pellet but still having problems checking that on electrophoresis.
We are trying now precipitating that DNA again with NaAc and ethanol. Do you know if repeated precipitations would further clean the DNA and get rid of polysaccharids?
Posted 21 February 2005 - 06:50 PM
Oh, and don't block the column with herring sperm DNA or such.
Repeated precips don't usually help. If you do your precips at room temperature, and only let sit for a few mins, then most polysaccharids won't have the chance to come out of solution.
Posted 24 February 2005 - 05:50 AM
I am out of ideas. Can anyone else help?
Posted 24 February 2005 - 08:47 AM
Posted 24 February 2005 - 11:53 AM
this isn't really a good reply cause i don't kow how to improve purification, its just that i have noticed with my own preps (bacterial genomic dna, yes even for us life isn't always golden) that some "tough" restriction enzymes will digest well DNA which most do not touch, for me EcoRV, DraI and RsaI ate it up, so maybe you can just get around your quality problem...
Posted 01 March 2005 - 12:54 PM