The DNA pellet that just won't resuspend - DNA extraction Large Scale (Aug/08/2005 )

Hi All,

I have been having trouble with my plant genomic DNA extractions. The organism is Momordica cochinchinensis and the plant material is the red fleshy seed coverings (arils) as opposed to leaves (The fruits is al I have access to). I crush the material in liquid N2 and incubate with CTAB with 2% pvp and 5% beta-mercaptanol at 65C. I use phenol:chloroform:isoamyalcohol (25:24:1) followed by chloroform:IAA (24:1) extractions until the interphase is clear. DNA precipitation is with 1M NaCL and cold isopropanol and then a 70% ethanol wash. The problem is that no matter how much water I try to resuspend it in, the pellet just won't go back into solution. Jabbing it with a pipette tip reminded me of sticky hair gel. Help please I really need high quality concentrated gDNA to even begin my project. Sorry about the realllly long post too.

Phil

some additional info:
Phenol is buffer saturated with 10mM Tris HCL pH 8.0
Spec of water after a weekend of trying to resuspend pellet:
concentration (A260-A320): ~100-190ug/mL
quality ratio ((A260-A320)/(A280-A320)): ~1.8-2.0

Hi Phil,

Do you need RNA? An RNAse treatment should get rid of some of the sticky-ness. Also try incubating it overnight at 4 C (though if you've been working on it over the weekend, you've probably done this anyway). One of the (many) gDNA protocols I have for plant DNA extraction has the final resuspension in R40 buffer (40ug/ml RNAse A in 1 x TE), which you leave it in overnight at 4 C. Sometimes I like to incubate it on a gentle rocking platform too, I don't know if that helps, but it gives me the illusion that I'm doing something while it's just hanging around in the cold room

Whatever you do, do not vortex it! It will shear the DNA.

I also find that freezing it at -20C then thawing it on ice can sometimes help with the resuspension, though you don't want to do it more than necessary (see above re: shearing).

How long is your 70% EtOH wash? Sometimes increasing that will help too.

And don't underestimate the amount of DNA you have. The spec reading wont be accurate until the DNA is in solution - better to resuspend it in a greater volume, find out that it's too dilute then re-precipitate it than to mess about with it at an unknown concentration.

Good luck,

Nicole

Hi, Phil,
I worked on seaweed and I used to faced the same problem. As I know so far, the "gel" feeling is not caused by RNA (I used to treat the extract with RNAase but no use). It 's due to polysaccharide. Actually I am also looking for a better protocol to remove those polysaccharides. Perhaps you can take a look on some papers in the journal "XX Plant Reporter" (Sorry, forget the exactly name). For that journal I found pretty many papers talking about DNA extraction methods in fruit plant. Hope this inforamtion is helpful. : )

It is almost certainly polysaccharides that are your problem. Are you vacuum drying your pellets? If you are then don't - it is much easier to resuspend pellets if they have been allowed to air dry only (10 min with the lids off should be enough).

Daniel

Better DNA sequencing

Duh! Sorry about that Phil! I had a blonde moment.

Polysaccharides can be removed using the relevant parts of the following method: Chomczynski, P. and Mackey, K. (1995). Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19:942-945.

I use the following modification of this (as found in Suzuki, Y. Hibino, T., Kawazu, T., Wada, T., Kihara, T. and Koyama, H. Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate. Biotechniques 34(5): 988-993.) for removal of polysaccharides:

Add a 1/3 x volume of 1.2M NaCl-0.8M sodium citrate and a 2/3 x volume of isopropanol. Incubate at room temperature for 10 min. Precipitate by centrifugation, wash with 75% ethanol twice, air-dry and resuspend in TE buffer or water.

The references are just in case you want to look them up

Good luck,

Nicole

huhuhu

After browsing the forums for bit, I decided to go check my latest sample (2in50 dilution):
A260 = 0.2737
A280 = 0.1631
A230 = 0.5609
A320 = 0.0087
(A260-A320)/(A280-A320) = 1.71
(A260-A320)/(A230-A320) = 0.479
conc (A260-A320)*25*50 = 331.25ug/mL

As you can see my A230 reading is crazy, which means I must still have a lot of phenols in it. But isn't 2% pvp and 5% beta-mercaptanol supposed to deal with the phenols? I have a feeling that my A230 reading is so high that my A260 reading isn't accurate

Will that 1/3 x volume of 1.2M NaCl-0.8M sodium citrate remove the polysaccharides better than NaCL alone? I've read that some people use an Acetate because they are easier to remove if u don't want a high conc of salt in your DNA.

Anyway according Fred_33 post:
http://www.protocol-online.org/forums/inde...pic=4476&hl=230
I've got to find a way to bring my A230 down... alot...

Phil

ps. Thank you for all the suggestions, they were really helpful

I can't recall the exact reference, but I ran across a journal article describing genomic DNA extraction from maize seeds. They must have had to deal with polysaccharide reduction/removal if the isolated from seeds, so you might want to check that out.

-Hank

hi phil

In large genomic extracxtions u generally have a large pellet so it can be difficult to resuspend all of it. I had the same problem when i was working with brassica.

1. If u are having a problem with phenol (i dont like it at all) try using dual phases like isoamyalcohol and chloroform. easier to handle and generally gives very pure DNA.

2. As far as the pelleting is concerned, avoid vacuum drying and decrease ur spin times with ethanol washes(2-3 mins usually sufficient to give a stable loose pellet). I usally find pellets with longer centrifugations really tough to resuspend

3. TE buffer is better than water to store the DNA

4. Dont worry about dissolving the whole pellet as for most molecular analysis u would not use morte than 10% percent of ur prepared sample. The clear liquid would have enough DNA.

Hope this helps

Prajwal

OK... I'm going to try it again Friday, this time without the Phenol:chloroform:IAA extraction and just the chloroform:IAA. I'm also goin to decrease the starting material from 1g to 0.5g per tube as well. I'm using 50mL falcon tubes by the way and all the spin are done at Rm temp to keep the CTAB in solution. Still haven't figured out a way to get rid of polyphenols except for the pvp and the antioxidant beta-mercaptoethanol. I've heard of a rumor that if u don't get rid of the polyphenols during the extraction they will irreversibly bind to your DNA. Is that true? I haven't come across anything like that in the literature. Does that mean that my current gDNA sample is screwed? can someone more experienced than me clarify this. Here's a thought... maybe I should ask a chemist to see if they know how to get rid of polyphenols without harming my DNA

Phil

Hi Phil,

I should have said before, but I'm working on eucalyptus and they have loads of polyphenolics and polysaccharides.

One method I'm using for RNA extraction I have mentioned above (the Suzuki paper) and their answer for polyphenolics is to use a strong reducer along with the B-mercapto. They (and I) use Sodium D-isoascorbate at a conc of 500mM in the extraction buffer (500mM SIA, 100mM Tris-HCl, pH8.0, 10mM EDTA, 5% b-mercapto, 2% SDS).

I'm absolutely sure that the polyphenols attach irreversibly to the NA (I'm working with RNA, but DNA would be the same) since no matter how hard I try with RT-PCR on samples extracted using other methods I can't get amplification. This includes the RNEasy qiagen column! The SIA method is the most reliable that I have used (although the most stinky because of the mercapto!)

I can forward you the article if you like.

Nicole