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southern size standards

southern size standards - Protocols and Methods Forum

southern size standards - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 06-25-2008, 07:38 PM
Ed Siefker
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Default southern size standards



I'm looking for a DNA size standard I can use with southern blots. I don't
want to deal with radioactive gel apparatus, so I don't want to end label
my ladder before I run the gel. The ideal solution would be something dyed
so I could see with the naked eye on the membrane, and mark the sizes with
radioactive ink. Is there something like this available?

Here's an idea, I can see the DNA ladder in my gel under UV. Could I inject
a small amount of P32 into each size standard, and then transfer so the size
standards would be visible on the membrane?

I've seen protocols for making size standards from lambda DNA, but these
require probing with lambda DNA. If I do this, would I include the lambda
probe in the hybridization with my probe? Do I need to reprobe with the
lambda probe?

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  #2  
Old 06-26-2008, 12:03 PM
peter
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Default southern size standards

On Jun 25, 3:38 pm, Ed Siefker <[Only registered users see links. ]> wrote:

Just stain the membrane with ethidium Br or other fluorescent dye.
After the transfer you usually can see your ladder quite well. In
Southern (depending on the complexity) it is difficult to avoid seeing
the ladder due to non-specific hybridization .
my2c
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  #3  
Old 06-26-2008, 12:03 PM
peter
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Default southern size standards

On Jun 25, 3:38 pm, Ed Siefker <[Only registered users see links. ]> wrote:

Just stain the membrane with ethidium Br or other fluorescent dye.
After the transfer you usually can see your ladder quite well. In
Southern (depending on the complexity) it is difficult to avoid seeing
the ladder due to non-specific hybridization .
my2c
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  #4  
Old 06-26-2008, 12:03 PM
peter
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Default southern size standards

On Jun 25, 3:38 pm, Ed Siefker <[Only registered users see links. ]> wrote:

Just stain the membrane with ethidium Br or other fluorescent dye.
After the transfer you usually can see your ladder quite well. In
Southern (depending on the complexity) it is difficult to avoid seeing
the ladder due to non-specific hybridization .
my2c
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  #5  
Old 07-03-2008, 10:23 PM
Jose de las Heras
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Default southern size standards


"Ed Siefker" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ].n et...


I tend to simply add a *minute* amount of the ladder to my probe during the
labelling reaction. Then you see the ladder as well as your probe... but of
course, you have to be sure there won't be crosshybridisation.

Jose


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  #6  
Old 07-04-2008, 09:51 AM
Kay Schink
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Default southern size standards

Ed Siefker schrieb:
In our lab, we have frequently observed cross-hybridisation of labelled
probes with the DNA Ruler Ladder Mix from Fermentas (no affiliation).
Interestingly, this happens only with this marker, but not with others
like Lamba-Pst - so you could give it a try if this wouks with your
probes, too. We also usually photograph our gels with a ruler next to
it, so by measuring the distance from the wels to our band, we can
interpolate the sizes.

Good luck

Kay


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