Restriction digestion giving extra bands

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nanda
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Restriction digestion giving extra bands

Post by nanda » Thu Nov 15, 2007 5:22 pm

I'm working with a pUC19 based plasmid. My gene of interest is inserted in the EcoR I site of pUC19. I tried to cut out my gene with pvu II enzyme. I should have got bands at 5.9 and 2.3(2 bands only)..but i'm getting bands at 1.7 and 2 regions also. It can't be due to star activity as it is said that the buffer i use don't give star activity. What could this be due to...?Yea.. and i loaded 2 micrograms(10microlitres) of the plasmid..enzyme used was 0.6 microlitres(10U/microlitre). Digestion was for 6 hours.

blcr11
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Post by blcr11 » Thu Nov 15, 2007 5:44 pm

So you have an insert of about 5.4 kb with two PvuII sites separated by 2 kb? There are no PvuII sites in pUC19 so the 2 kb fragment can only come from the two internal sites—at least I think so. How certain are you that you really do have only 2 PvuII sites? Is there any chance that your tube of enzyme is contaminated with a small amount of a different restriction enzyme? Those are the only things I can think of off the top of my head: more than two PvuII sites, or possible contamination.

nanda
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Post by nanda » Thu Nov 15, 2007 5:53 pm

Thanks for the reply benny..But there are 2 pvu II sites in pUC19..and none in my insert.. I'm positive of that..Could it be that my 2.3 bands got resolved separately..? and it's not possible that there was some other enzyme contamination coz i'm getting 5.9+2.3= 8.2 which is the size of my palsmid..... :?

blcr11
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Post by blcr11 » Thu Nov 15, 2007 6:41 pm

Sorry. I just quickly looked at a map, which evidently only showed the once-only cutters. I didn't check the full list of sites. Looks like I shoud have. I still can't think of any explanations other than more sites or contamination, though.

ali12345
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Re: Restriction digestion giving extra bands

Post by ali12345 » Wed Jun 03, 2015 9:38 pm

nanda wrote:I'm working with a pUC19 based plasmid. My gene of interest is inserted in the EcoR I site of pUC19. I tried to cut out my gene with pvu II enzyme. I should have got bands at 5.9 and 2.3(2 bands only)..but i'm getting bands at 1.7 and 2 regions also. It can't be due to star activity as it is said that the buffer i use don't give star activity. What could this be due to...?Yea.. and i loaded 2 micrograms(10microlitres) of the plasmid..enzyme used was 0.6 microlitres(10U/microlitre). Digestion was for 6 hours.



I know this was posted years ago, but I am having the same exact problem. Did you ever figure it out?

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