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Tuesday, April 5, 2011

Fwd: gel electrophoresis resolution



---------- Forwarded message ----------
From: Cory Tobin <cory.tobin@gmail.com>
Date: Tue, Apr 5, 2011 at 2:18 AM
Subject: Re: gel electrophoresis resolution
To: diybio@googlegroups.com


I've never actually seen a gel quite like this one.  The tailing of
the bands is usually due to loading too much DNA in the well.  But the
bands don't look that bright.  Maybe there actually is a lot of DNA
there but since the background light isn't being filtered out it just
doesn't appear very bright?

Some general gel troubleshooting:

- Make sure the the loading buffer is the same as the running buffer.
For example, if you used 1X TAE in the gel and you are using a 5X
loading buffer, the loading buffer should be made with 5X TAE so when
it's diluted in your sample the pH and salt concentration is the same
as 1X TAE.

- Load less DNA

- Replace the running buffer, assuming you have been reusing it
multiple times.  Not as critical if you're using TBE.

- Use a lower voltage.

- Fill the chamber lower.  5mm over the top of the gel is ideal.
Excessive buffer leads to smearing.


For more gel debugging and optimization check out a pdf called "The
Sourcebook" by the company Cambrex.  http://tinyurl.com/3pqbts8


-Cory

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