SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
How do i get rid of Formamide from my dsDNA? mzahir89 Sample Prep / Library Generation 5 10-08-2015 04:05 AM
Phenol-chlorophorm DNA extraction samani_nima Complete Genomics 1 05-08-2013 04:18 AM

Reply
 
Thread Tools
Old 02-17-2017, 02:41 AM   #1
SeqTroubles
Member
 
Location: Ireland

Join Date: Sep 2016
Posts: 19
Default Phenol-chloroform ext and degraded dsDNA

Hi All,

I have been trying to extract high quality dsDNA for NGS using phenol-chloroform methods, with and without CTAB and different lysis buffers.
But I always get a huge discrepancy between nanodrop and qubit (dsDNA kits) readings. I recently measured my extracts with the ssDNA kit and found the majority of my DNA (>90%) is degraded to ssDNA.

The DNA is from Cronobacter species with ~52-56% GC, grown overnight (~18hrs). I have used the JGI CTAB protocol and standard phenol-chloroform extractions with SDS lysis buffer. During separations I do not vortex, just invert the tubes several times with ~2sec of a vigorous handshake. The pH of the phenol is stated as >7 but I did measure it with a pH strip which indicates its 7 and could be possibly be slightly less. The phenol is not buffered with tris.

Thanks for the help
SeqTroubles is offline   Reply With Quote
Old 02-17-2017, 04:55 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,237
Default

Some possibly useful info here.

Briefly, one issue is that even trace amounts of residual phenol will confound attempts to assay DNA concentration using UV spectrophotometry (nanodrop). Second, genomic DNA preps always have large amounts of RNA in them. RNAse, by itself, does not change this -- it just converts the RNA into oligonucleotides and mononucleotides, both of which absorb UV just fine. Third, are you sure your ssDNA assay isn't sensitive to RNA?

--
Phillip
pmiguel is offline   Reply With Quote
Old 02-17-2017, 07:28 AM   #3
ATϟGC
Member
 
Location: Canada

Join Date: Jun 2013
Posts: 36
Default

If RNA contamination is your issue the following likely won't help you, but it might if you problem is contaminants:

For recalcitrant DNA extractions I highly recommend trying an CTAB dilution-based precipitation method such as those described in the following two articles:

Xin and Chen 2012:
https://plantmethods.biomedcentral.c...1746-4811-8-26

Arseneau, Steeves and Laflamme 2016:
http://onlinelibrary.wiley.com/doi/1...12616/abstract

I find these methods work very well if you have a sample with moderate to high amounts of DNA (such as fresh or well preserved plant and animal tissues). These methods might not work well with low-yielding or degraded tissues like archival plant samples (herbarium specimens). These methods give high yields of very pure DNA in my experience as they are based on a different precipitation principle (DNA-CTAB precipitation by NaCl dilution) than most traditional homebrew and commercial kits, which largely rely on alcohols and/or chaotropic salts to precipitate nucleic acids.

If your Cronobacter lyse well in CTAB some modification of CTAB-NaCl dilution should give you high yields of high molecular weight DNA.

If you have trouble accessing the second article and supplementary protocol you can request it from Royce Steeves on reserachgate.
ATϟGC is offline   Reply With Quote
Old 02-17-2017, 08:26 AM   #4
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,237
Default

I should also mention that the statment:

Quote:
I recently measured my extracts with the ssDNA kit and found the majority of my DNA (>90%) is degraded to ssDNA.
seems pretty unlikely to me to be the case. Unless you heated your DNA to above its strand-melting temp and the snap-chilled it, there isn't really a way to convert most of it to ssDNA.

Having an ssDNA assay that is sensitive to RNA also seems much more likely.

Oh, except that the most likely explanation is that 90% of the signal you saw in the nanodrop spectrum at A260nm was actually just the shoulder from trace amounts of phenol remaining in your sample. Take a look at your nanodrop spectrum -- if the peak is at 270nm, then that is phenol, not DNA/RNA.

--
Phillip
pmiguel is offline   Reply With Quote
Old 02-22-2017, 02:56 AM   #5
SeqTroubles
Member
 
Location: Ireland

Join Date: Sep 2016
Posts: 19
Default

Hi All,

Thanks for the suggestions. I have some tris buffered PCI incoming and I will let you know how it goes. In the mean time.

My latest back of results from an extraction by a colleague yielded the same results as follows:

nanodrop qubit BR dsDNA qubit ssDNA
W70 330ng/ul 30ng/ul 300ng/ul

Ive ran the sample overnight on 0.5% agarose and have a big bright band.
The nanodrop spectrum is peacking at 260nm.

I have done PCI extractions before and I have never had this issue. The tris buffer is the only thing I can think of at the rate.
SeqTroubles is offline   Reply With Quote
Old 02-22-2017, 06:25 AM   #6
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,237
Default

Quote:
Originally Posted by SeqTroubles View Post
Hi All,

Thanks for the suggestions. I have some tris buffered PCI incoming and I will let you know how it goes. In the mean time.

My latest back of results from an extraction by a colleague yielded the same results as follows:

Code:
          nanodrop        qubit BR dsDNA    qubit ssDNA
W70     330ng/ul            30ng/ul             300ng/ul
Ive ran the sample overnight on 0.5% agarose and have a big bright band.
The nanodrop spectrum is peacking at 260nm.

I have done PCI extractions before and I have never had this issue. The tris buffer is the only thing I can think of at the rate.
The ssDNA assay just isn't going to be accurate for a genomic DNA prep.
From the web-site:

Quote:
...nucleotides and short oligonucleotides of six bases or less do not interfere with the OliGreen ssDNA quantitation assay. However, the OliGreen reagent does exhibit fluorescence enhancement when bound to dsDNA and RNA.
Similarly on the qbit ssDNA assay website:
Quote:
The Qubit® ssDNA Kit is ideal for quantitating single-stranded DNA or oligonucleotides. However, it is not specific for single-stranded DNA. This assay kit will also detect double-stranded DNA and RNA but it will not detect contaminating protein or nucleotides.
So it looks like the signal generated by the Qbit ssDNA assay will be confounded by any dsDNA and any RNA in the sample. Genomic DNA preps will typically be >90% RNA unless heroic measures are taken to remove the RNA. Like RNAse digestion followed by a column. Although a highly effective mixture of RNAses might degrade RNA below the 6 base threshold of Qbit ssDNA/oliGreen quantitation.

--
Phillip
pmiguel is offline   Reply With Quote
Old 02-24-2017, 07:50 AM   #7
SeqTroubles
Member
 
Location: Ireland

Join Date: Sep 2016
Posts: 19
Default

So I have just performed the extraction with fresh tris buffered PCI and its the same issue. I have a 2hr lysis incubation with RNase A prior to PCI extraction and the agarose gels do not indicate the presence of RNA.

Ill run a restriction digest next week to determine whether this is a ssDNA issue.
SeqTroubles is offline   Reply With Quote
Old 02-25-2017, 09:22 AM   #8
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,237
Default

Quote:
Originally Posted by SeqTroubles View Post
So I have just performed the extraction with fresh tris buffered PCI and its the same issue. I have a 2hr lysis incubation with RNase A prior to PCI extraction and the agarose gels do not indicate the presence of RNA.

Ill run a restriction digest next week to determine whether this is a ssDNA issue.
Do you do any high temperature incubations? I can't think of any other way you might be ending up with large amounts of ssDNA.

Also, keep in mind that RNAseA, by itself, is probably not going to be able to degrade all the RNA to less than 6 base oligonucleotides. So your ssDNA assay is probably just detecting RNA degraded small enough that it doesn't show up on a gel (0.5% agarose -- you probably can't resolve anything below 100 base on that.)

You would need to do a column clean-up -- like the genomic DNA one that Zymo offers -- to get rid of the RNA oligos. Or take some other heroic action to remove that degraded RNA.

We used to hook the genomic DNA out of solution using pasture pippets and then soak them sequentially in a couple of tubes with 1 ml of 70% ethanol, before transferring the clump of genomic DNA to a microfuge tube and spinning/pippeting off the residual ethanol.

But for many uses, the DNA doesn't need to be that free of RNA.

--
Phillip
pmiguel is offline   Reply With Quote
Old 02-28-2017, 03:36 AM   #9
SeqTroubles
Member
 
Location: Ireland

Join Date: Sep 2016
Posts: 19
Default

Hi Philip,

Thanks for your help. I did a digest with xbaI with and ran a neat undigested sample also. The genomic DNA was undigested and the neat sample has a lot of RNA despite best efforts. So I think Ill stick with my kit extractions for now.

Regards
SeqTroubles is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 09:50 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO