Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Methods for desalting


  • Please log in to reply
43 replies to this topic

#16 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 16 July 2010 - 09:07 AM

Why dont you test the 260/230 ratio if the samples are rich of humus?

If the ethanol is cold when you add to your samples its ok.
I never centrifuge my samples in a fridge or a centrifuge you can refrigerate.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#17 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 16 July 2010 - 09:49 AM

I am. The A260/A230 is around 0.10 to 0.15. Shouldn't it be >1.5?

I found a refrigerated centrifuge in my biophysics neighbor's lab :lol: .
Still waiting for my sodium acetate and I'll roll.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#18 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,617 posts
127
Excellent

Posted 16 July 2010 - 09:59 AM

If you still have a humic acid problem this paper might help:
D. Sutlovic, M. Definis Gojanovic, & S. Andelinovic: Rapid Extraction of Human DNA Containing Humic Acid.
Croatica Chemica Acta 80 (1) 117-120 (2007).
They added polyvinil-polypyrrolidone to avoid TaqPolymerase inhibition.
(The names are a bit different with Slavic letters, I cannot write here)

A single lie is reproachable; a million lies is a statistic.
D. J. T.


#19 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 16 July 2010 - 10:32 AM

I am. The A260/A230 is around 0.10 to 0.15. Shouldn't it be >1.5?

I found a refrigerated centrifuge in my biophysics neighbor's lab :lol: .
Still waiting for my sodium acetate and I'll roll.


didnt you mention it was the a320 that was about 0.15?


Well good luck with it and the paper Hobglobin mentions, might be helpfull.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#20 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 16 July 2010 - 11:00 AM

Oh this is funny. I read that paper yesterday. It's interesting, they show that humic acids have an impact on several kind of enzymes. However, they don't suggest anything to get rid of them :lol: .
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.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#21 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,617 posts
127
Excellent

Posted 16 July 2010 - 11:07 AM

Oh this is funny. I read that paper yesterday. It's interesting, they show that humic acids have an impact on several kind of enzymes. However, they don't suggest anything to get rid of them :lol: .
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?

A single lie is reproachable; a million lies is a statistic.
D. J. T.


#22 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 16 July 2010 - 11:09 AM

didnt you mention it was the a320 that was about 0.15?


Yes between 0.15 and 0.003.
Do you know what the numbers should be for A260/A230 and what they mean? Protein contamination?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#23 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 16 July 2010 - 11:12 AM

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?


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.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#24 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,617 posts
127
Excellent

Posted 16 July 2010 - 11:36 AM

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?


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.

A single lie is reproachable; a million lies is a statistic.
D. J. T.


#25 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 16 July 2010 - 11:51 AM

A260/A230 above 1.8 ??? :lol:
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.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#26 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 17 July 2010 - 02:20 AM

didnt you mention it was the a320 that was about 0.15?


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)

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#27 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 20 July 2010 - 05:27 AM

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.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#28 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 21 July 2010 - 02:30 AM

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?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#29 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 21 July 2010 - 07:20 AM

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?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#30 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 21 July 2010 - 07:45 AM

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..

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.