In researching why one would get streaks down the lanes instead of discrete bands, the only suggestion I have found is that there is too much DNA. In my case, I am sure this is not the reason, having gotten good results with some of the strains I am currently testing. Any ideas?
Hi Candace, have a look at the web site www.pulsenetinternational.org/.../pfge/PFGE_troubleshooting.pdf
There are many ways to avoid smearing. Good luck
smeering could be due to:
-one of your purification steps (avoid any harsh mechanical step)
-high level of impurities (check protein/DNA/carnbohydartes percents)
No pics - I don't see the need to record the failures. The bugs are Bordetella, prepared by embedding in agarose plugs, lysing with lysozyme, followed by overnight in proteinase K. Plugs are washed for hours to rid of protein fragments, then digested with restriction enzyme. I have used both XbaI and ApaI with similar results.
Smearing could be also caused by cutting your PFGE slices too thick so that your restriction enzyme doesn't cut your DNA properly. Also if you don't wash all the PMSF from your slices this could inhibit your restriction enzyme causing smearing.
You can also check the PFGE troubleshooting section in the CLSI guideline MM11-A.
I have observed streaking due to current fluctuations working with staph...
Thank you for all your suggestions. However, I got the solution from Sonali Mahalanabis, tech rep at bio-Rad. She that smearing usually indicates DNA degradation from contamination of the tubing or electrophoresis chamber, and suggested replacing the tubing and flushing the system for 2 hrs with a fresh 10% bleach solution, followed by clean water rinse. That did the trick.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology