8 replies to this topic

[planty]

Hi all!

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?

Thanks!

[leahf]

Hi
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.

[bob1]

Hi

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
Bob

Edited by bob1, 20 February 2005 - 07:49 PM.

[planty]

Hi ! Thanks for your replies

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?

Thanks!

[Great White Northern]

I have had many problems isolating plant genomic DNA because of contaminants such as the ones you mention. Right now, I am in the process of trying to column-purify my DNA. I use a sephadex G50 column (the same kind we make for separating probes from free label). If your DNA solution is too viscous, it may take a few spins to get the DNA out. If so, you probably carried a lot of salt, and some phenolics along in the second spin. Make a fresh column, and repeat if this is the case. If your sephadex is stored in TE, then calibrate the column using low TE. Today was actually the first day that I tried this. The tan colour is gone from sample, and the A260/A280 is fine. I did take about a 25% loss on my DNA. Tomorrow, I will be doing a restriction digest to check restrictability and flourescence. I will post the results.
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.

[Great White Northern]

Unfortunately, the column purification was not that successful. My HindIII digests look a bit better, but my EcoR1 and BamH1 still look undercut.

I am out of ideas. Can anyone else help?

[Great White Northern]

Sorry to be flooding the replies, but upon closer examination of my gels, I think the column purification did help out more than I had originally thought. I think this method shows promise, but I'm not yet sure how to refine the process. Perhaps someone has some suggestions.

[leahf]

Hi Great white Norhthern
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...

[Great White Northern]

Thanks, leahf, but I also have methylation issues to contend with. This really limits the enzymes I can use. Plus, I usually need 6 base cutters. However, if you have realiable, methylation-insesitive enzymes that you use, I would love to hear about them.