Preparative acryl amide gel electrophoresis

Gys de Jongh GysdeJongh at csi.com
Fri Jun 11 17:12:59 EST 1999


Raja Kota <rkota at scu.edu.au> wrote in message
news:rkota-0806991636320001 at ip3956.scu.edu.au...
> Hi Netters,
>
> I have been cloning some AFLP bands from polyacrylamide gel.  Once I
> excise the band from the gel, I resuspend it in sterile water (50µl) o/n
> at 37C.  Following this I use part of this as my template and re-amplify
> the fragment.  However, every time I re-amplify the fragment, I do not get
> the expected size.  Intrestingly all the bands are approximately half the
> size of the original band.  For instance, if I had excised a 500 bp
> fragment, I seem to re-amplify a 250 bp fragment.  I am bewildered by this
> observation and am looking at some way to explain this phenomenon.  If you
> have any suggestions, please e-mail me directly on rkota at scu.edu.au
>
> Thanks in advance,
>
> Raja

inderpal singh <ips98 at HOTMAIL.COM> wrote in message
news:19990610160651.90999.qmail at hotmail.com...
> Dear Netizens,
>     I am encountering a problem while trying to isolate an AFLP band of
size
> ~250bp from the AFLP ladder i generate in a 12% acrylamide gel. The
problem
> is when i excise the band, perform a re-pcr and load it again to check, i
> see, alongwith my desired band, a couple of bands greater and smaller than
> it, that have presumably got amplified.     This happens even when i cut a
> tiny area of the desired band to eliminate contaminating bands.
>    Please tell how to get rid of the undesired bands so that i can clone
the
> band of my interest.
> Thanks in advance,
> IP


Liz Parks <liz_parks at ncsu.edu> wrote in message
news:37611EFB.1D7084C4 at ncsu.edu...
> I have seen the same thing happen with AFLP bands of all different
> sizes.  However, when I load this reamplified product on an agarose or
> mini NON-denaturing acrylamide gel, I only see a single band of the
> correct size.  Go ahead and clone your product.  When you sequence it,
> you will most likely get a fragment of the correct size.  I did.  I
> don't know what causes the extra banding on the denaturing PAG .  Could
> be denaturation of secondary structure?
>
> Good luck!
>
> Liz
>
> --
> **********************************************
> Liz Parks
> North Carolina State University
> Department of Plant Pathology
> Box 7616
> Raleigh, NC  27695

Hi,
we are trying to manipulate dna which has been isolated repeatedly from
acryl amide gels in the sage technique http://www.sagenet.org/
1) re-amplifying 1 band , cut out of acryl amide , by pcr often gives more
than one product (both larger and smaller)
2) RE digests are always partial.
3) cloning of this dna in a plasmid and transformation is not very
succesfull ; small number of colonies.

If the bases are chemically modified than this would explain the inhibited
enzyme reactions.
In standard organic text books it can be found that activated amides react
with any basic group. We mixed acrylamide/bis-acrylamide with lambda dna :
longer exposure=>less digestion. We found a number of articals where the
carcinogenic properties of acrylamide are correllated with dna alkylation.
In one artical this was via the glycylamide (acrylamide oxidised by oxygen
radicals) intermediate.

Could it be that the dna reacts chemically with the acryl amide monomer ?
or with the oxygenradicals from the persulfate/temed ?
or with the glycylamide formed by the above two ?
Has anybody used acryl amide gels for preparative purposes ?
Thanks for the input
Gys de jongh







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