Help: Blotting DNA from acrylamide gel

Hiranya Roychowdhury hroychow at NMSU.EDU
Tue Jul 18 09:53:12 EST 1995


 On Tue, 18 Jul 1995, Stuart Brown wrote:

> This is a real stumper for me.  I need to make a Southern blot of some
> PCR products after separating them by size in a polyacrylamide gel.  I
> need to use acrylamide in order to resolve small size differences between
> products (2bp SSR alleles) and I need to probe in order to verify that
> my PCR products really contain the repeats that I'm supposed to be
> amplfying.  According to "Current Protocols" you can't use simple
> capillary transfer on polyacrylamide, they reccomend electroblotting.
> I found an electroblotter (Pharmacia "Multiphor II") and gave it a try
> using my "best guess" conditions (0.5X TBE buffer, 200 mA, 20 min) with
> absolutely no useful result (ie. blank film after probing filter and
> long exposure). I called the Pharmacia tech help line and was told that
> they don't have a reccomended protocol for blotting DNA with their machine
> because nothing that they have tried worked very well.  Come on, this has
> got to be possible!
>  
> Does anyone out there have any experience blotting DNA out of acrylamide?
> No reasonable suggestions sneered at.
>  
> Cheers - Stuart Brown
>  
> Stuart Brown                       |   Plant Genetic Resources
>                                    |   Georgia Experiment Station
> INTERNET:                          |   1109 Experiment Street
>   SBROWN at GAES.GRIFFIN.PEACHNET.EDU |   Griffin, Georgia 30223-1797 USA
> BITNET:   SBROWN at GRIFFIN           |   Fax: 404-229-3323
> 
> 




The first thing that comes to my mind is that the transfer time was too
short. Also, try reducing the TBE to 0.25x and do the transfer, if
possible, in an ice bath in the cold room. 

Here is another possibility: vacuum suction transfer. I am not sure if
such a set-up exists in the market such a transfer. But if your gel fits
in one of the regular gel driers then you can use that. Place the matrix
(NC or Nylon) on a sheet of 3MM, and place the gel on the matrix. Now
cover them up with Saran wrap and the rubber/silicone flap (that is
supposed to be used with the drier) in that order and apply vacuum without
any heating. If this works, the transfer time has to be worked out
empirically. I wonder if denaturing the DNA in situ would help in such a
transfer. 
	The premise of my suggestion is that unless fixed to the gel by
some means, proteins in gels are not retained following drying in the
above manner. 
			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
			<<<<<<<<<<<<<<<<<<<<<<<<<<<




More information about the Methods mailing list