Methods for desalting - (Jul/14/2010 )
Maddie on Jul 16 2010, 09:00 PM said:
.We all want to get rid of them but they bind to silica, they seem to precipitate during an ethanol prec...they are really a bother. When working with ancient DNA, you don't want to make too many handy work because of the contamination issues. So most protocols that could help remove the HA aren't really recommended for people working with small amounts of human DNA. I found a very interesting paper on HA yesterday where they show how to comfirm the presence of HA with fluorescence. Since my buddies in biophysics have the instrument, maybe I'll try to measure what's left in my extracts.
I guess the humic acids bind to the polyvinil-polypyrrolidone and are then removed or inactivated.
BTW what about alternative methods like CTAB or chelex for DNA extraction? Can't they remove those humic acids?
pito on Jul 16 2010, 02:32 PM said:
Yes between 0.15 and 0.003.
Do you know what the numbers should be for A260/A230 and what they mean? Protein contamination?
hobglobin on Jul 16 2010, 03:07 PM said:
BTW what about alternative methods like CTAB or chelex for DNA extraction? Can't they remove those humic acids?
CTAB, PTB and Chelex have been quite unsucessful. I had read about the polyvinil-polypyrrolidone and then forgot about it. I will google this again. The problem with HA is that it is a mixture of different kinds of molecules with different size and proprieties. I wish I had a filter with a 500bp cut off so I would concentrate my extract and keep what goes through instead of what's retained.
Maddie on Jul 16 2010, 09:12 PM said:
hobglobin on Jul 16 2010, 03:07 PM said:
BTW what about alternative methods like CTAB or chelex for DNA extraction? Can't they remove those humic acids?
CTAB, PTB and Chelex have been quite unsucessful. I had read about the polyvinil-polypyrrolidone and then forgot about it. I will google this again. The problem with HA is that it is a mixture of different kinds of molecules with different size and proprieties. I wish I had a filter with a 500bp cut off so I would concentrate my extract and keep what goes through instead of what's retained.
well they are successful against a range of organic molecules especially from plants and often a remedy if organic acids (e.g. tannic acids) or polysaccharides inhibit reactions...surprised that this methods won't help here.
Anyway polyvinilpolypyrrolidone should do the job, if the paper is correct.
For interpretation of the A260/A230 this paper might help.
A260/A230 above 1.8 ??? 
Wow I am VERY far from that. Maybe because I have so little DNA, my A260 is really low and the contaminants don't help. That sucks.
I also thought HA had to do with polyphenol but here is what I read yesterday (and bear with me, I'm not biochemist)
Humic acids originate from polysaccharides containing xylose, arabinose, and fructose. Xylose and arabinose oxidize and polymerize via the furfural and 4-oxo-2-butenoic acid pathway, and fructose oxidizes and polymerizes via the 5-hydroxymethyl furfural and 4-oxo-2-butenoic acid pathway.
Maddie on Jul 16 2010, 09:09 PM said:
pito on Jul 16 2010, 02:32 PM said:
Yes between 0.15 and 0.003.
Do you know what the numbers should be for A260/A230 and what they mean? Protein contamination?
Arent you mixing up the a320 and a230 ??
because in your first post you speak about the a320 is 0.15 and then you mention its the 230 thats 0.15 ?
and you use the 260/230 ratio if the samples contain many humus like products/organic products
(like allready mentioned in the paper and it should be around 1.8 or even better 2.0)
I measure absorbance at 230, 260, 280 and 320mn.
320 for the turbidity; I guess humic acids would affect that.
A260/A280 for DNA purity but I know that some molecules from the "humic acid soup" will mess up the A260 as well as the A280.
So basically, none of my measurement is where it should be and the humic acids (or/and the salts?) are responsible. I still don't know how to know how to assess what comes from the HA and what is caused by the salts.
I have always been told that 230 is used if you work with samples with a lot of humus
280 if the sample is "clean"
320 for turbidity.
Anyway, your results arent really good.
Have you decided to do an ethanol precipitation or what will you do now?
I did. Only 1 sample out of 3 ended up with a good ratio of A260/A280.
I unfortunately think I pipetted the ethanol on the wrong side of the tube, so I'll try again.
My quantities of DNA are super low though and I'm wondering if this couldn't also explain the bad ratios.
Waiting for qPCR data on the 3 extracts I cleaned.
What should 230 be when there is lots of HA?
Eum it could indeed be that you pipetted the wrong side of the tube. But as you also stated: you had not much DNA.. this could also be the reason.
Anyway, I always had good results with ethanolprecipitation, but you need to be carefull and maybe add 2 samples toghether before you start the precipitation. And make sure not to spin down to much before using the nandodrop.
What the value should be when there is a lot of HA? Dunno, I only keep in mind that a good ratio for 260/230 is 1.8-2.0.
If the value is rather low it means there are a lot of contaminants, so you value means there is a lot in your sample with a max absorbance at 230... humus, polysaccharides..