Hi,
I am trying to characterize some MRSA strains with PFGE. The DNA marker (SC DNA ladder or Lambda DNA ladder) migrates nicely through the agarose gel. The bacterial DNA on the other hand does not. I see the DNA stacked in the well. I am not sure whether the bacterial genomic DNA is digested or not. I tried several protocols but were not successful. Any recommendations? Should the genomic bacterial DNA migrate in the gel even if it is not digested?
Undigested bacterial genome would not run on PFGE. You should try to follow change in the composition of your lysis buffer (from any other lab optimised procedure).
It could be due to defective restriction enzyme too.
Try to use a new vial of restriction enzyme.
Hi,
Thanks for all of you for your help and sorry because I could not reply earlier.
I had also the same thoughts. It could be either ineffective lysis of the bacteria or not digested DNA. To exclude the lysis problem, I extracted bacterial DNA using Qiagen DNA extraction kit from bacteria invoving lysis with lysozyme. Then perform SmaI digestion. I got the same results, a bright band in the well, although the marker is migrating nicely. Then I used SmaI enzyme from another company (NEB, Invitrogen) and in the last attempt i also used HindIII enzyme additionaly. But unfortunately I got the same results. The enzymes are working fine because they can digest a small plasmid containing an SmaI site. What could be the reason for undigested genomic DNA other than the quality of the enzyme? I already use high quantity of the enzyme.
HI Khaled
I have done PFGE for Streptococcus pneumoniae. What I usually do is to embed intact bacterial cells in soft agarose plugs followed by lysis of the cells. After bacterial lysis I proceed with the enzymatic digestion. This is also done for MRSA.
Check out this paper: http://www.ncbi.nlm.nih.gov/pubmed/18025669
Good luck.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA