smearing in bands after digestion

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smearing in bands after digestion

Post by Newbie » Fri Jun 02, 2006 9:00 pm

Hi everyone,

I was wondering why I keep seeing a smear in my bands after digestion of my vector. I digested with 1ul EcoR1 and XhoI in a 50ul reaction for 2-3 hours. I also ran the uncut vector along side it and noticed that the smear appears only after digestion. My post-doc told me to just cut the bands out and avoid the smeared parts but the fact that there is a smear after my bands bother me. Anyone know why and should I be concerned about it?

tranjo
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Post by tranjo » Sat Jun 03, 2006 3:08 pm

If you're band is clean, and migrates at the expected, then you shouldn't worry. I think it's common phenomena if you digest alot of dna, and run it on a gel, your bands sometimes look like they're "on fire." I've gel extracted the "core band" as well as the "flames" and they turn out to be identical (except if your digestion is incomplete). Gel extract it and run an aliquot on another gel.

BTW, I don't think there's an explanation for the migration anomaly. It happens even if you have pure sample resuspended in water. It's probably related to how much DNA you load on.

Best, J

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Post by Newbie » Mon Jun 05, 2006 2:57 pm

My band does migrate to the expected size but I know if I can call it clean since there is a smear...but my uncut vector doesn't have a smear though

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Post by dae » Mon Jun 12, 2006 12:57 am

It shouldn't be an issue for EcoRI (I'm not as sure about XhoI) but I had a similar problem when I let my restriction digest go too long - the enzymes started cutting unspecifically resulting smear below my desired band. I redid it for 1 hour _at_ 37 C and the smear disappeared. It's worth giving a shot. (I was also doing 50uL reactions).

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