degraded DNA in PFGE - (Nov/10/2006 )

Hello everybody!

I am having problems with my PFGE protocol.
I work with Enterococcus strais, and I am able to extract the DNA from the strains (wich was suposed to be the hardest part). However, the DNA presents degraded after the running time.

does anybody here could help me?
thanks a lot.

Are you sure its degraded and not just sheared? What are you trying to do with the DNA? Are you wanting to size the chromosome? Your DNA extraction protocol is likely to cause DNA shear resulting in a smear when you run it on PFGE. This is due to vortexing and pipetting steps. If you want to prevent this then the only way is to set the cells in an agarose block and lyse the cells and extract the DNA while in the block. The block is then loaded into holes made in the PFGE gel. The protocol takes a couple of days since you need to incubate the agarose containing cells with buffer for longer periods of time.

Degradation of genomic DNA during PFGE analysis did not occur exclusively during DNA isolation. I agree care should be taken during DNA preparation in order to preserve it as intact as possible. ML1975 is right with this respect.
Freeze/thawing of samples may affect DNA – avoid it or add EDTA.
Also, nuclease contamination of the gel-loading buffer - could give a false indication of DNA degradation.

But the DNA was shown to be also degraded upon the formation of free Tris radicals during the electrophoresis. This could be prevented by the addition of a free radical scavenger, particularly thiourea, to the running buffer. If so, DNA degradation should occur independently of the particular bacterial strain you are working with, during all DNA electrophoresis procedures in which a Tris buffer is used (clearly not the case). What then? Another component -most likely a protein, partially resistant to PK digestion - must be involved. This should be present in bacterial strains in which degradation of DNA occurs during the standard PFGE procedure, and it should be absent in strains in which DNA degradation does not occur.
For example, if degraded DNA is detected at the first plug assessment (without heating), then the cleanliness of the materials used to generate the plugs and the thoroughness of the washes should be considered. Poor PK digestion allows traces of cellular endonucleases that degrade the DNA. If heat degrades DNA in a piece of the gel slice, then the preparation and running of the preparative gel and the isolation of the gel slice should be evaluated (to avoid exposing the gel, running buffer, beta-agarase, cutting tools, etc. to EtBr, and the gel to UV).
A picture would be more suggestive still

I do want to size the microorganism’s chromosome! I know that the protocol used in this methodology causes a share in the DNA, which is what I really want to see!! I do set the cells in an agarose block, lyse it and extract the DNA in the block. The fact is that there´s no shear, just “smear”!!!

Hello

here I am aganin!
now I'm having another problem wich can, in part, be associated to the first one.
The degaradation I was talking about before was due to buffer's contamination, and fortunatelly, now I'm not having this problem anymore...

The problem now is that I'm using a pulse marker (0,1kb a 200kb from Sigma) and having problems with the gel, wich do not presents the marker very well...