E. coli and SDS-PAGE

Allison Haggarty ar229 at FreeNet.Carleton.CA
Fri May 20 08:59:17 EST 1994



In a previous article, mkertesz at micro.biol.ethz.ch (Michael Kertesz) says:

>In article <bushnt.1119742558A at usenet.rpi.edu>, bushnt at rpi.edu (Timothy Bushnell) says:
>>
>>
>>    In an undergraduate lab, we are having the students prepare protein
>>profiles of a stationary phase E. coli culture using SDS-PAGE. 
>>
>>    The problem is that the sample is very viscous and near impossible to>successfully load on a gel.  Typically, after the sample is loaded into the
>>wells, and the pipet tip removed, the sample sticks to the tip and is
>>removed as a gob into the upper tank buffer.. 
>>
>
>If you sonicate the extracts briefly, it should be possible to break the DNA
> in the extract into small enough bits that the viscosity problem is 
>overcome. A few seconds with a standard sonication tip should do the
>trick. Foaming shouldn't be too much of a problem with this volume. Addition of DNase
> is also a good (and quick) method, but has the drawback, in principle, that you
>are adding an additional protein to a mixture where you wish to analyse the
>protein composition, and thereby introducing an unnecessary artefact. In practice. however, the amount 
>of DNase added can be so small that you can't see it, especially if you are using
> Coomassie Blue staining to visualize the proteins.
>
>Michael Kertesz
>
>ETH Microbiology
>Zürich, Switzerland
>

When we are making RNA we also want to get rid of the contaminating DNA. 
We break it up by simply pipetting up and down until the solution is no longer
viscous.
-- 
Allison Haggarty          ar229 at freenet.carleton.ca
                          mdah at musica.mcgill.ca




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