smear after restriction digestion

[Fanch]

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?

[javaid]

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

[canalon]

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

[wangc]

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!!!!!!!!!

[wangc]

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.

[wangc]

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.

[sinadas]

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.

[JackBean]

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.