2 replies to this topic

[ocean_sky83]

Hi

Has anyone encountered a problem with pulse field gel electrophoresis on bacteria and ends up with a smearing at the lower molecular weight bands. Does anyone knows how to troubleshoot this?

I read about Tris break down causing free radicals during electrophoresis.. Beside using thiourea to scavenge the free radicals, is there any other alternatives?

Thanks!

[phage434]

The use of thiourea is only needed in certain species that phosphothionate their DNA.  You have not said what your pulse times are.  Normally, resolution is established at different length scales by control over the switching times of the pulse.  See any manual on pulse gels, e.g. the Biorad ones, or almost any article for details on the choice of switching times.

[hsb31]

ocean_sky83, on Mar 24 2010, 07:08 PM, said:

Hi

Has anyone encountered a problem with pulse field gel electrophoresis on bacteria and ends up with a smearing at the lower molecular weight bands. Does anyone knows how to troubleshoot this?

I read about Tris break down causing free radicals during electrophoresis.. Beside using thiourea to scavenge the free radicals, is there any other alternatives?

Thanks!

The best way to get PFGE is to play with pulse and cells amount. First standardize pulses for your Mb sizes using a PFGE marker, vary the agarose percentage also to get the right resolution. If cells are lysed completely and restriction digested, it will resolve in nice bands, just try to increase the amount of cells.