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

#31 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 21 July 2010 - 09:14 AM

Here is what I got for 3 extracts (ethanol precipitated if I may say that) that all gave DNA concentrations under 5ng/ul

Sample 1:
A260/A280: 1.3
A260/A230: 0.1

Sample 2:
A260/A280: 1.11
A260/A230: 0.23

Sample 3:
A260/A280: 1.85 :D
A260/A230: 0.08 <_<
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

#32 pito

pito

    Veteran

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

Posted 21 July 2010 - 09:19 AM

what was the DNA concentration prior to ethanolprecipitation?

Those results do suck lol

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


#33 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 21 July 2010 - 12:28 PM

No, this is after the ethanol precipitation. BUT it was even worse before :lol:
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

#34 pito

pito

    Veteran

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

Posted 22 July 2010 - 01:51 AM

Yeah, I know, but did you measure it before?
Did you measure the DNA quantities before eth.precipitation? You need to do that.

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


#35 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 22 July 2010 - 07:35 AM

Oh yes I had

Sample 1:
A260/A280: 1.05
A260/A230: 0.15

Sample 2:
A260/A280: 0.95
A260/A230: 0.13

Sample 3:
A260/A280: 1
A260/A230: 0.14

And for DNA concentration:
sample 1: 5.3 to 1.5 (ouch)
sample 2: 3.9 to 4.3
sample 3: 4.7 to 2.3

Here is what I read about the nanodrop:

The manufacture-stated dynamic range for the NanoDrop
instrument is large: 2 - 3,700 ng/μl of dsDNA. In practice,
GenVault finds that DNA concentrations of less than 10
ng/μl are seldom reliable as the basis for A260-based
DNA concentration determination (see Fluorometric
measurements section and Table 3 for explanation), and
DNA concentrations of less than 20 ng/μl are seldom reliable
as the basis for the more demanding analysis of DNA purity
via A260/A280 or A260/A230 ratios.

This could explain my bad ratios maybe.
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

#36 pito

pito

    Veteran

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

Posted 22 July 2010 - 07:39 AM

Yeah, you are working with small amounts, so its hard.

But to be honest, what I would have done is added the samples together.

And the first one, that you lose so much DNA is strange.

PS. you didnt really improve a lot ... its sounds strange to be honest.
What protocol did you use for ethanolprecipitation?

Edited by pito, 22 July 2010 - 07:42 AM.

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


#37 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 22 July 2010 - 08:10 AM

Here is the protocol I used (found on Bioforum :) )
I don't have glycerol unfortunately.

50ul of extract
+ 130ul cold Ethanol 100%
+ 5ul Sodium acetate 3M pH 5.2
Mix (I didn't dare to vortex. Ancient DNA is damaged enough already)
Place in -80C Freezer for 2 hours
Centrifuge for 30 min. at 4C (more than 12,000g)
Remove alcohol without disturbing the pellet. :P

Add 200ul of 70% ethanol (-20C) and centrifuge for 5 -10 min at 4C.
Pipette out all the remaining alcohol
Centrifuge briefly for a few seconds and pipette out all the remaining liquid.
(Let it dry with open lid)
Add warm TE/water and wait for 2-5 min. and then mix and centrifuge.

For sample 1, I probably pipetted most of the pellet.
I will try again with other samples.
I could pool several extracts of a sample together and that would increase the A260. I'm afraid it would also increase the A230 a lot though.
Next week I will try this protocol I mentioned on the other posts and will clean up DNA with silica beads. I hope it will help.
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

#38 pito

pito

    Veteran

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

Posted 22 July 2010 - 08:18 AM

How do you remove the ethanol?

Do be honest: I would certainly not pipette out the alcohol in the second stage... NEVER

And why do you need the glycerol?

I never use that.

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


#39 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 22 July 2010 - 09:01 AM

Yes, I confess father, I did use the pipette (bad Maddie). Although in my previous life, I was just flipping the tube. I guess that's how you do it, right?
OK, OK, I will start all over again with new extracts.
The glycerol is supposed to help when you precipitate very small amounts of nucleic acids (I read that here as well).

Sooo what's the magic trick to quick a.. with an ethanol precipitation? :P
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

#40 pito

pito

    Veteran

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

Posted 22 July 2010 - 09:19 AM

Eum I can see why you add the glycerol (I have seen that here too) but I never did it.

Anyway: like you said: just put the tube upside down on a sheet of paper to let it dry. Let it stand upside down for maybe 40 minutes, then flip it and let it stay for another 40 minutes or so.
(before flipping it back , make sure to remove the moisture at the side of the tube (the drupplet of fluid thats hanging there.. if not, even better)

And the magic trick is to be very cautious. And before you measure the DNA on the nanodrop: do not spin it down. You might spin down the DNA so much that when you measure you dont measure it since you only pipetted water or whatever...

and when you see you are low on DNA after the first step (before the eth. precipitation) you might wanna add 2 samples together.

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


#41 mdfenko

mdfenko

    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,383 posts
220
Excellent

Posted 22 July 2010 - 12:13 PM

shouldn't that be glycogen rather than glycerol?

talent does what it can
genius does what it must
i used to do what i got paid to do


#42 pito

pito

    Veteran

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

Posted 23 July 2010 - 03:48 AM

shouldn't that be glycogen rather than glycerol?


You are right mdfenko, its glycogen, not glycerol.

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


#43 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 585 posts
20
Excellent

Posted 23 July 2010 - 05:36 AM

I googled this product, concerning humic acid removal. I have never used this kit before. It and other similar products might be of interest to this discussion.
May your PCR products be long, your protocols short and your boss on holiday

#44 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 23 July 2010 - 07:15 AM

Oops, sorry guys, you're right: glycogen (which I don't have either anyway <_< ).

Perneseblue, funny you mention this kit. I have a student working on bacteria from soil extracts and she has a similar problem as me. The HA inhibit her qPCR. She uses this MP Bio kit to extract and let me tell you that it does not remove all the humic acids. I mean it worked for most of them, but not all.
Some of her samples were really bad and had a A260/A260 lower than 1.
Yesterday she tried 3 clean-up methods on the worse samples. First she extracted some samples in triplicate. Then she either did 2 independent clean up with the MinElute Qiagen kit (do the whole precedure, then all over again with a new column). 2nd method, she did phenol-chloroform followed by MinElute and finally 3rd method: MinElute and ethanol precipitation. They all gave similar results. Tiny bit better but the A260/A280 was 1.2 for all samples.
When she did the ethanol precipitaion, she could see a yellow pellet :( . So HA does indeed precipitate with the DNA, and it is water soluble so can't be extracted with phenol-chloro-isoamyl and the cherry on the pie, HA bind to silica.
Gosh, are they annoying :angry:
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




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

©1999-2013 Protocol Online, All rights reserved.