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smear after restriction digestion

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Postby Fanch » Fri Apr 20, 2007 3:57 am

Yes sure the problem might come from me and it was that I thought until someone else in the laboratory has the same problem…
And we used different buffer and water etc.. so I did not contaminate these one in a preceding manipulation...
Anyway, what kind of error I'll have been able to make to contaminate all my tubes each time?
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Postby javaid » Fri Apr 20, 2007 1:00 pm

u may try by incubating the sample without buffer...if it gets smeared then the problem is with enzyme...as per u both the mini n midi prep had the same results then it is the thing that is common in both isolations n that hopefully is only the enzyme so u may try with other enzyme ...... hope this helps
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Postby canalon » Fri Apr 20, 2007 7:52 pm

What I meant is that it can be a problem with the manufacturer. And the complete Buffer Lot might be contaminated, so even if you use a different tube from the same lot you still have the same contamination.

As javaid suggest if the contamination comes from the enzyme you have to reorder some. But before doing that can you test with a few µl borrowed from another lab. If all your buffer tube have the same lot# you can also roam your university/institute for helpful people with some buffer to spare (more likely than enzymes too).

Good luck
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Postby wangc » Thu May 17, 2007 4:07 am

Help!!!
I got the exactly same problem.
I did the BamH I single digestion for two plasmids. one of them perfectly ok, but the another one totally smeared. since I used the same buffer, enzyme, etc, to digest the two plasmids, only one have problem. so the digestion system should be ok. I regrow the bacteria and use a new Qiagen miniprep kit to prepare the plasmid, and digested again, the problem presisted.
so I trying to find to what is the problem. I incubate my plasmid with water only, BSA only, BUffer only and Buffer plus BSA respectively. after overnight incubation, the water and BSA one give beautiful 3 bands plasmid, while the Buffer and Buffer+BSA gave nothing, looks like all the plasmid has been degraded.
so it looks like the problem is the buffer I used. however, I used this same buffer to digest another plasmid, it is totally ok.
big confuse.
is there one possible reason that there are some contamination in the plasmid prepration. And this contaminated enzyme can only function in the digestion buffer?

Help!!!!!!!!!
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Postby wangc » Thu May 17, 2007 4:23 am

I think think and think.
this might be due to the Ecoli strain I used, which is sbtl3.
several months ago I used this Ecoli to extract the another plasmid, but have the same problem. I just ignored it at that time since I later used maxi prep to prepare the plasmid and then no problem.
so might be there is something in the Ecoli that miniprep kit extraction cannot remove it, while the endo-free maxiprep can remove it. I will test it tomorrow by extract the plasmid with endo-free maxiprep and then digest it, to see what will happen. I will tell you the results ASAP.
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Postby wangc » Fri May 18, 2007 8:29 am

I used the Qiagen endo-free maxiprep to extract the plasmid today. I then did the enzyme digestion, and the digestion is perfect. clearly one band.
so the problem is this specific strain of Ecoli, sbtl3, which may contain something cannot be removed by normal mini prep kit. using endo-free kit can remove it and get normal results.
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Re: smear after restriction digestion

Postby sinadas » Tue Oct 06, 2009 4:31 pm

Hi,
I did a digestion using EcoRI and NcoI restriction enzymes. I wanted to digest my plasmid (5 kbp) but when I saw the gel I just obtained a smeared band. I don't know why because the gene that I wanted to insert was cut perfectly in the same conditions. My boss told me that this could be due to the strain I used to extract more plasmid, but... Now I'm going to transform my purified plasmid in other cells and I'll repeat the digestion. What would you do to solve this kind of problems??? Do you know why it happens??? I don't think this is due to DNases or another contaminant.
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Postby JackBean » Wed Oct 07, 2009 6:17 am

Hi all,
I'm a little bit confused by "normal 3 band pattern" in your runs, because I've always seen only 2 bands in the uncut plasmid.
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Postby Eddie » Tue Jan 19, 2010 8:29 pm

I have had the same problem - a smear which occurs only after incubation with restriction enzyme buffer, but which isn't present in the plasmid itself, and which isn't a problem for other plasmids when they are incubated with the same tube of restriction enzyme buffer.

The problem looks like contamination of the plasmid by a nuclease which is inactive but which is activated when it has access to some components of the restriction enzyme buffer (eg. Mg2+). I'm still trying to find out possible causes of nuclease contamination (I've had variable results using the same kit) - right now my major suspect is whether I'm talking while doing the miniprep (i.e. breathing on the sample too much) :?

JackBean, about the "3-band pattern" that plasmids give on a gel - apparently it can be up to 5 - take a look at this: http://en.wikipedia.org/wiki/Plasmid#Conformations
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Postby JackBean » Wed Jan 20, 2010 12:06 pm

Yeah, but usually, when you have intact plasmid, you should have only relaxed and supercoiled circular DNA, shouldn't you?

Did you try to incubate the plasmid with full reaction except the enzyme?
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Re: smear after restriction digestion

Postby kimsmarkin » Wed Sep 22, 2010 7:09 am

The problem is that Earl plasmid isolated from the same team that gives reasonably good results, but after digestion of the last two defamation preparation for.I think it is nuclease contamination.
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Postby jasen » Sat Feb 05, 2011 12:38 pm

Hi everybody, Im getting problem with restriction digestion of plasmid DNA as Im getting smeared band, on digestion of plasmid with EcoRv....Plz help soon
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