I am currently working on PFGE for P.aeruginosa . I use SpeI for the restriction. The bands obtained are always blurred/ smeared. • I wash the plugs thoroughly after lysis step. • I carry out restriction for 4 hrs. • I have tried 50uM of Thiourea .Electrophoresis conditions and instrument - CHEF DR III apparatus (Bio-Rad) at 14 °C, 6 V/cm, for 20 h, with a time switch of 5–30 s. also PFA for recent gel pic
Dear Upasana, I think your cell concentration is too high. For this reason they couldn't lyse completely. You can decrease cell amount, lyse them in plugs and maybe increase the amount of restriction enzyme. Usage of thiourea prevent degradation of DNA in running gel. Be sure the temperature of buffer (14C).
Good luck.
mert
Mert Sudagidan Can any one tell me why my band is not go down after running 24 hour 6 volt (5 to 20 second) in PFGE test for bacteria Acinetobacter baumannii ?
Dear Nazmul, in my opinion, DNA was not cut by restriction enzyme in left plugs. In right plugs, bacteria is not digested completely and not bacterial DNA cut by restriction enzyme. You can try to prepare your plugs again to increase your bacterial digestive enzyme like lysozyme etc. and cut again by restriction enzyme. Another posibility your enzyme could not work well, maybe you can change it.
Best regards.
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