smearing in bands after digestion? - Why does a smear appear after digestion? (Jun/02/2006 )
Hi all,
I was curious to know why I keep seeing a smear after my bands after digestion of my vector. I ran the gel of the uncut vector along side with my cut vectors which I digested with 1ul of EcoR1 and XhoI in a 50ul reaction and I notice that my cut vectors always show a smear that is not seen in my uncut vector. Why is that? My post-doc told me to just cut the bands and avoid the smear but the fact that there is a smear bothers me.
I was curious to know why I keep seeing a smear after my bands after digestion of my vector. I ran the gel of the uncut vector along side with my cut vectors which I digested with 1ul of EcoR1 and XhoI in a 50ul reaction and I notice that my cut vectors always show a smear that is not seen in my uncut vector. Why is that? My post-doc told me to just cut the bands and avoid the smear but the fact that there is a smear bothers me.
why don't you show your gel here to see the smear????
I guess your plasmid DNA gets degraded during the incubation time of your restriction digestion. Your uncut vector looks ok because you just took several ul directly from the tube. I would either purify the plasmid DNA again a/o use a new aliqout of enzyme, buffer, water etc. to get rid of DNAses.
Yes, I agree with chalet2 -- your plasmid is being degraded, either by contaminating nucleases or by star activity.
Run a few different controls to find out what's going on -- do a mock digest of your plasmid, where you prepare the "digest" the same -- use the same water, same 10x buffer, same incubation time, but no enzyme -- just add additional water equal to what the enzyme amount would have been. Also incubate the aliquot of your uncut plasmid DNA alone at 37°C, to see if the nucleases are active in your plasmid prep. I would also do single enzyme digestions, to see if one or the other enzyme is to blame.
In general, though, the post doc is right -- if you get good bands of appropriate length on top of fainter smearing, you can cut these out of the gel and proceed.
Run a few different controls to find out what's going on -- do a mock digest of your plasmid, where you prepare the "digest" the same -- use the same water, same 10x buffer, same incubation time, but no enzyme -- just add additional water equal to what the enzyme amount would have been. Also incubate the aliquot of your uncut plasmid DNA alone at 37°C, to see if the nucleases are active in your plasmid prep. I would also do single enzyme digestions, to see if one or the other enzyme is to blame.
In general, though, the post doc is right -- if you get good bands of appropriate length on top of fainter smearing, you can cut these out of the gel and proceed.
Thanks for the suggestions!...I'd run these controls except I do not have much plasmid left, perhaps I should try to cut the incubation time shorter...they tell me 2 hours should be enough...