unsuccessful gel purification - (May/24/2007 )
hi all i amplified differnt promoter deletions i got the specific band after pcr then i sliced the target band to gel purify it but i got a result driving me crazy after gel purificatin i got my target band very clear and another longer band please sombody help me
Contamination in your elution buffer?
Sorry, only idea coming to my mind...
Reload the entire purification on a gel, run, cut and purify again to remove contaminants.
Run the gel slowly and then try to cut and purify the band. Somtimes if the gel is run too fast, they carry unwanted DNA along with them.
throw back the DNA on a gel and gel purify the DNA a second time.
As scolix has rightly stated, sometimes running the DNA too fast, causes other DNA run together with your desire band.
In addition, running too much or too high a concentration of DNA in too small a well, can also cause unwanted DNA to co-migate with your desired band.
But what is "to much" in do not run to much DNA on the gel?
I would say my maximum limit is 800ng per 1cm well width.
what you want in a gel purification, is a narrow straight neat DNA band. You don't want a big fat band.
One of my colleagues was too concerned about having a lot of DNA in a single well, he would run like 2-3 ug of DNA spread out in 10 wells. He would run them slowly overnight. He would cut out all the bands and purify the DNA over a few hours.
if you get your target band then you can ignore contaminant bands. and also as krumel said check your elution buffer