Help: Warped Polyacrylamide Gels

Beth Wapelhorst weazel at seanet.com
Sat Dec 3 21:57:57 EST 1994


hoelzer at unr.edu (Guy Hoelzer) writes:

>Hello MoBo Netters:

>We are trying to use the Penguin polyacrylamide gel apparatus from Owl to
>separate microsatellite PCR products in my lab.  We are running 1.5 mm
>thick, non-denaturing gels followed by staining with SYBR Green I.  The
>gels have been very warped with smeared bands that run very unevenly across
>the gel.  The only reason we are not running a standard sequencing gel is
>that it is much more convenient to use and stain the smaller (20x20 cm) gel
>from the Owl rig.  I would appreciate any suggestions that would help us
>achieve nice tight bands that run evenly across the gel.  Thanks in advance
>for your help.

Well, there's ten billion things that could be the cause of this. Here's 
a few possibilities:
1. Pouring the gels - it's really important to not let the gel set 
propped up with something under the glass plates. Prop it up by the edges.
2. Crappy acrylamide, dead ammonium persulfate, anything that keeps the 
gel from polymerizing all the way.
3. Non-denaturing gels have to run at a cool temperature, which means you 
have to run them slower than a denaturing gel or else they'll get too 
hot. This is the main cause of warped gels - uneven heating. Also, if there 
are air bubbles under the bottom edge of the gel, the electricity will 
arc and make parts of the gel REALLY hot. This is a good way to crack your
plates.
4. Your bands might be getting fuzzy from the staining process. It 
doesn't really take too long for the DNA to diffuse through the gel after 
the current is turned off, so if your takin' your time, the bands will 
really spread out.
5. 1.5 mm is a pretty thick gel, maybe you could try a 0.4mm one.
6. Non-denaturing gels usually give pretty globby bands for 
microsatellites anyways. Why don't you use denaturing ones?

Hope this helps.

sugar beth
weazel at seanet.com




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