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High background in Southern hybridization
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High background in Southern hybridization
I've had annoying problems with high background in standard Southern
hybridization. I have digested 5 ug of human genomic DNA w/ EcoRI, BamHI
and HindIII and used several different probes (500 bp, no Alus or other
repeats, labeling with Amersham RediPrime II), but the signal is always
very weak compared to the background smear. Hybridization is at 60-72C,
first wash with 2xSSC, 0.05% SDS at 65C.
I've used alkaline blotting (0.5M NaOH, 1,5M NaCl), hyb solution is simply
10% dextran sulphate, 1M NaCl, 1% SDS (works well with Northerns
etc.) with 0,15 mg/ml herring sperm DNA. Addition of Cot-1 DNA (1
ug/ml) does not seem to reduce background, commercial ExpressHyb solution
does not give any better results, either. Has anybody had similar
problems? Do hybridization solutions with formamide give usually any
better results?
I use charged Hybond N+, but I crosslink the DNA with UV, nevertheless.
Mikko Taipale
Newsgroup archive
I use Hybond also, and in my hands UV crosslinking increases the
background enormously. You might try simply baking to crosslink
instead and see if that helps your background problem.
Good luck,
John
Newsgroup archive
Do you also use charged nylon and alkaline blotting? With positively
charged membranes, I believe there is no need for additional crosslinking
provided that the blotting is done with an alkaline solution.
Mikko
Newsgroup archive
We get very good results Hybond N+ with alkaline blotting. As mentioned
by Mikko, cross-linking occurs during the alkaline blotting process. Two
not-so-intuitive things have been found to lower background in our hands:
1) Don't let the membrane dry between blotting and pre-hybridization. I
rinse 2x in 2x SSPE after blotting and then put the blot in a bag and
start the prehybridization immediately.
2) The capacity of the membrane seems to be extremely high, and you need
to use enough prehybridization and hybridization buffer to saturate the
non-specific binding capacity (ca. 0.1 ml per square centimeter). It's
necessary to use fresh buffer for the hybridization itself; don't just
add probe to the prehyb buffer. (I use buffer with 6X SSPE, 50%
formamide, 5x Denhardt's, 0.5% SDS, and 250 ug/ml denatured salmon sperm
DNA. I think hybridizing in plastic bags is superior to hybridization
tubes. Shaking or other movement during hybridization is unnecessary. My
final wash is with 0.5X SSPE--0.2% SDS in a plastic bag submerged in a
65 C water bath.)
Something that has also helped a lot (but not because of background) is
to use Kodak BioMax MS film and screen. The sensitivity is so high with
this combination that the bands are overexposed after 48 hours.
--
Ned Mantei
Newsgroup archive
I've gotten much better results by switching to the "Modified Church
and Gilbert Buffer" that Amersham recommends for Hybond
(0.5 M NaPO4 pH 7.2, 7% SDS (w/v), 10 mM EDTA) - and it is much
simpler to make up, as well.
John
Newsgroup archive
Is this the stuff Boehringer sells as Easy Hyb? It must be a high SDS buffer,
but it seems to me (my nose) that there is ammonia in it. Does anyone have a
recipe? Has anyone used home-made high SDS for Northerns?
--
Susanne Rohrer
Newsgroup archive
Yes... For years...with northerns and southerns... no salmon sperm dna
just sds and buffer.
Check out the recipes in the BioRad protocol for ZetaProbe. The recipes
also work for other membranes. It appears to be the Gilbert buffer.
Newsgroup archive
Agreed! I haven't mucked with salmon sperm DNA for about the last 9 years. A
Japanese post-doc in the lab started coming out with these pristine genomic
southerns and with "cross-examination" he finally showed me the old NEN GeneScreen
manual. This is was around 1992 so I have no idea where NEN was getting/making
their membranes then. However, I do know as of this week that the manual has
changed and the box the membrane came in definately says "Pall" on the outside.
In short, what the "old" manual says was (paraphrased) that if the SDS is taken to
at least 1% in the hybridization solution then salmon sperm DNA isn't needed.
Whether it be normal or genomic southerns, northerns, or library screening, I
haven't needed ssDNA for the last 9 years as I've kept the SDS at no less than 1.2%
(the extra for insurance!).
I've never played with dextran sulfate either. When asked, many years ago my
post-doc mentor told me in no certain terms that dextran sulfate was a recipe for
high background and not to use it.
Hope this helps,
David
Newsgroup archive
> Do you also use charged nylon
Lately I've been using Hybond XL.
> and alkaline blotting?
Yes.
> With positively
> charged membranes, I believe there is no need for additional crosslinking
> provided that the blotting is done with an alkaline solution.
That's probably true, but I still bake them anyway (after neutralization).
Its the UV crosslinking that seemed to cause the background problems;
baking doesn't seem to do that (in my hands anyway).
John
Newsgroup archive
the Biorad's zetprobe protocol works out well, and I feel is more easier,
faster and cleaner than the traditional SSC method.
jayakumar
Newsgroup archive
Last update 21-Dec-2001, Rating Fair of 1 votes.
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I have a lot of hybond XL membrane and was wondering if I could use it for southern blots as well instead of ordering the hybond N+ that the protocol recommends. Does anyone know what the difference is?
Rating: n/a
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I am having problems with my hybridization experiments. I used to get quite good signals. But recently I am not getting. I have not changed any of my buffers or reaction conditions. Only my probe I have changed. Then the amount of DNA that has been blotted on to Hybond N+ membrane is 20 microgram. So the question of using less DNA can be eliminated. Regarding the probe I prepared it fresh many a times and checked on gel and found to be quite intact. Can it be that it is a low copy sequence or anything like that ? Somebody please provide an answer.
Rating: Good
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Problems with DIG Southerns...
(I) About Easy Hyb
DIG Easy Hyb - At a guess, that ammonia comes from 6M urea used to lower the hyb temperature...and at another guess, I'd put the SDS content at around 7-10% judging by viscosity....and I expect its around 1mM for EDTA, and buffered by phosphate (0.25M)...pH 7.2. It costs more than ?00 per 500mL here in the UK, so, if someone wants to try out the recipe above before I get a chance, email me the results!
Now, as for those tiny spots, they are supposedly caused by antibody precipitate, so you are recommended to spin down at 14,000 rpm for 10 minutes at 4 celsius (but no cooler) before removing your 3 microlitres per 30mL antibody.
Furthermore, the Roche blocking solution is far better for background than the one that is described in the manual, unless you change it to 3M NaCl 100mM free maleic acid pH8. Regardless, it still fails to adequately buffer once you add your 2.5mL per 50mL 10% blocking solution (which should be filtered before use thru miracloth)
The unincorporated DIG needs to be removed by a column, I'm trialing APBiotech GFX...
Never put Mg in the detection buffer...it precipitates arghhh in solution and on the blot presumably giving background...and finally...
Read:
"Detection of Single-Copy Genes in DNA from Transgenic Plants by Nonradioactive Southern Blot Analysis" by McCabe et al, Molecular Biotechnology, Vol 7(1), 1997, pages 79-84
best wishes,
Jon Rees
Webmaster for http://molbiol.net (Portal for Bioinformatics)
About my DIG troubles:
Yes, I am struggling to obtain superclean Southerns using DIG with B.napus and B.oleracea right now - the genome being 12 times the size of Arabi, its about the same as doing single-copy Southerns...but once you do this, you can do anything! I have formerly got it to work by buying in the Roche blocker (readymade solution), but I am too proud to give in at the moment...I have a basic protocol outlined here, but I will update it when I find one that works with 2 micrograms gDNA.
http://www.r9corporation.fsnet.co.uk/Methods/dignorthernf.htm
Rating: Good
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I have a problem with my hybridizations too. I get a lot of tiny block spots on my membrane. I have increased the amount of my wash buffers and I have used sterile containers for washing.
Could anyone help me?
I use a roller bottle and alkaline phosphatase detection kit. I always use fresh buffers and I even centrifuge the hybridization buffer before use to remove precipitates.
Rating: Good
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Does anyone know whether its necessary to bother RNasing plant genomic DNA before doing a Southern? Is it an unecessary step, only easing quantification? I'd like to hear from anyone who has tried this without RNasing...
Rating: Good
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