Originally Posted by swagemann
I'm having major problems with our PFGE. We've had Bio-Rad's CHEF DRIII setup for almost 10 years now. Our main project is to compare E. coli from a host animal species and compare it to a water sample to determine if there is any human dna found in the water body.
We were funded for 8 years by various entities, and the funding dried up. After 2 years we were contacted to run some samples. For the past year, I have not gotten any decent results. I've made up the reagents in our protocol 4 or 5 times, and even tried using Bio-Rad 'store bought' chemicals, and nothing seems to work. Here is what we know: we don't think it's the equipment. Lambda ladder control plugs are coming out fine. We don't think it's in the restriction enzyme step. We had an old Bio Rad JM107 E. coli plug (they no longer make it) and used our restriction enzyme onit, and it came out fine. There are times when it just looks like the lane is smeared. Mostly it looks like we get decent bands towards the top of the gel, but the bands then get progressively worse as we go down the gel.
Any one have any ideas?
OK. I cannot say that I am an expert but currently I am dealing with standardization of PFGE protocols for typing various nosocomial pathogens. We use Gene Navigator System (Amersham, now GE).So far I've made two gels (about 30 strains)- the first was fine (except that the time was too much) ...the second not so well, but acceptable. Maybe this is the newbie luck but here are some things I can say for sure. The cell concentration is extremely important.....0,8 to 1 OD measured at 600-610nm seems to work best. Do you have lysozyme step prior to prot K? How many times, at what temp and with what buffer do you wash samples after lysis. I wash 3 times with 2ml/2 blocks per tube TE at 4C, 1/2h each and then 100mcL restriction buffer for 1/2h at 4C; change restric. buf. and then do the restriction in 100mcl fresh buffer.
Do you add thiourea in the TBE? Check the literature- it helps alot...
As you see there are alot of factors contributing to failure ....that's why I truly hate this technique...besides there are so many other more discriminative and far more cheap methods. But all is a matter of economical interests....