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#1 Fredo

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Posted 24 January 2012 - 02:08 AM

Hi erveryone,
Currently, i'm trying to do some southern to check the number of copy of my transgene. I'm working on wine. After having loads of trouble to get rid of polysacchardides and polyphenol during dna extraction, i'm struggling now with the southern.

I'm working in a small lab and we don't have a lot of facilities and in this case we don't have a stratalinker to crosslink dna on the blot... Thus, i'm trying to do the crosslink with our geldoc. Do you think it's possible or the geldoc is not powerfull enough, because i've got no signal after revealing my southerns...

My other question is, how to force all the digested dna to run in the agarose gel. Because there's a lot of dna remaining in the wells after running...

Thanks for your help

#2 phage434

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Posted 24 January 2012 - 05:27 AM

Your gel doc system will not crosslink effectively, since it likely has a 305 nm UV source. The stratalinker uses 254 nm UV, much more effective at trashing DNA. You can probably bind your DNA to the membrane with heat, however. I believe some people autoclave southerns, but I'd probably start with a dry heat. Careful with nitrocellulose, which is extremely flamable.

You probably want to work your detection from the end towards the beginning. Spot serial dilutions of your DNA or a control on a membrane, and practice your detection. If you are using a probe, start by spotting serial dilutions of the probe, and detecting it. Then, serial dilutions of your sample, detecting with your probe. Only when that is working, start doing gels and gel transfers.

Long DNA fragments don't move from the well, so likely your DNA is not completely cut. This is probably not a significant problem.

#3 David C H

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Posted 24 January 2012 - 07:29 AM

The DNA may not be migrating because of polysaccharides. If you are sure the DNA is clean (260/230 absorbance > 2.0) then you probably have incomplete digestion, suggested above. DNA and polysaccharides can form a viscous complex that won't migrate through the gel. If the DNA solution is at all viscous, this is why the migration is poor.

#4 Fredo

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Posted 31 January 2012 - 05:53 AM

Thanks for the tips ! One more question, what type of agarose are you using ? In my we've only got a cheap one which is not very define for sizes larger than 2-3 kb..... we are a poor lab Posted Image

#5 David C H

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Posted 31 January 2012 - 06:44 AM

We don't use special agarose for southern blots. If you need separation above 3kb, run a lower % gel and run it long. We typically use an 0.8% gel run overnight at low voltage. You will need long gel apparatus for this (gel tray of 30cm or more). If you know your bands are larger than 3 kb, you can run the lower fragments off of the gel.

Edited by David C H, 31 January 2012 - 06:52 AM.


#6 phage434

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Posted 31 January 2012 - 06:49 AM

David, I think you mean a 0.8% gel. Even lower percentages work, but they become very fragile.

#7 David C H

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Posted 31 January 2012 - 06:52 AM

yes -- sorry about the mistake -- I have corrected it.




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