smear after restriction digestion

[javaid]

my plasmid prepration gives a total smear following restriction digestion.. what may be the possible reason.... need help

[SororSaudade]

probably DNA degradation

[Vagabond]

Have you run a sample of your purified Plasmid on the same gel? Is it smeared also or do you get one, two, three or more bands??
What method are you using to prep your plasmid?

If the plasmid prep is good, most likely a contamination of one of your solutions (buffers, water, etc) or enzyme stock. Open new ones.

Note you should always run a small sample of uncut (modified) plasmid on you gels right next to you ladder.

[javaid]

i isolated my plasmid by sigma mini prep kit. uncut sample runs fine and gives 3 bands but once i digest it with enzyme it gives smear more just icubating with buffer n without adding any enzyme it also leads to smear,, i think there is somewhere nuclease cintamination .. can anyone help where it could have originated n also can i somehow purify my sample...

[Vagabond]

Your sample should be fine for you stated that when run uncut you get your typical 3 bands. With a smear forming when you incubate with your buffer alone, sounds like this is your most likely source of contamination. Get a new aliquot of buffer. Where it came from hard to say: How old is it? Who has used it? Would be the two biggest questions.

[javaid]

actually the problem is that earleir plasmid isolations by the same kit give quite good results after digestion but the last two preprations give the smear... so i think it is nuclease contamination ... but now i need to purify that sample n get rid of nucleases from that sample .is it somehow possible.....

[Vagabond]

If you newly prepped plasmid is degraded when you run it on a gel I would think that it is all degraded by this time. I would have to say there is a problem with your kit and that you will need to re-grow and re-purify. I am a big fan of Qiagen if you lab will buy it for you.
GL

[javaid]

actually it gets smeared only during restriction digestion coz when i run uncut sample it gives good bands but gets smeared after restriction digestion n even if i dont add the enzyme n incubate only with buffer... so i think it is nucleases which get active in buffer... now i need to purify my sample of those nucleases.. plz devise any method....

[SororSaudade]

does the restriction enzyme you're using have star activity? It could be cutting where it shouldn't.

[javaid]

no i does not have star activity whereas my earlier preprations of plasmids give good results after using these enzymes...

[Fanch]

Hi all !
I just "UP" this topic because I've the same problem..

So, I want to digest my plasmid with Pst1 and Sal1 ( I use NE Buffer 3)
After digestion my plasmid gets smeared, whereas my uncut plasmid gets the 3 "normal" bands.
I thank the problem could be a star activity, so I tried with less enzyme according to NE book. Moreover I tried with just one enzyme and plasmid gets smeared.
I thank that it could be a problem of nuclease contamination, because when I incubate my plasmid with just the buffer it gets smeared. So I tried with an other buffer, new water, and BSA. Same problem !
Finally, I thank that it could be my plasmid prep ( I use a Qiagen miniprep kit). So I tried with another plasmid from a Midi prep. Same problem..
Moreover, another co-worker in my Lab have the same problem and we don't use the same electrophoresis or pipette or tips...
But we use the same ladder, so I will try with a new ladder.
It's really strange !
Thanks a lot for your help ! ... and sorry for my english (I'm french)

Fanch

[canalon]

I do not think it can be your ladder, Fanch, because it should never be in contact with your digestion.
But your results are weird, but did you check if your 2 tubes of buffer belong to the same lot? The problem might not come from you...