smear after restriction digestion

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    • #6999
      javaid
      Participant

      my plasmid prepration gives a total smear following restriction digestion.. what may be the possible reason…. need help

    • #69293
      SororSaudade
      Participant

      probably DNA degradation

    • #69302
      Vagabond
      Participant

      Have you run a sample of your purified Plasmid on the same gel? Is it smeared also or do you get one, two, three or more bands??
      What method are you using to prep your plasmid?

      If the plasmid prep is good, most likely a contamination of one of your solutions (buffers, water, etc) or enzyme stock. Open new ones.

      Note you should always run a small sample of uncut (modified) plasmid on you gels right next to you ladder.

    • #69372
      javaid
      Participant

      i isolated my plasmid by sigma mini prep kit. uncut sample runs fine and gives 3 bands but once i digest it with enzyme it gives smear more just icubating with buffer n without adding any enzyme it also leads to smear,, i think there is somewhere nuclease cintamination .. can anyone help where it could have originated n also can i somehow purify my sample…

    • #69373
      Vagabond
      Participant

      Your sample should be fine for you stated that when run uncut you get your typical 3 bands. With a smear forming when you incubate with your buffer alone, sounds like this is your most likely source of contamination. Get a new aliquot of buffer. Where it came from hard to say: How old is it? Who has used it? Would be the two biggest questions.

    • #69382
      javaid
      Participant

      actually the problem is that earleir plasmid isolations by the same kit give quite good results after digestion but the last two preprations give the smear… so i think it is nuclease contamination … but now i need to purify that sample n get rid of nucleases from that sample .is it somehow possible…..

    • #69383
      Vagabond
      Participant

      If you newly prepped plasmid is degraded when you run it on a gel I would think that it is all degraded by this time. I would have to say there is a problem with your kit and that you will need to re-grow and re-purify. I am a big fan of Qiagen if you lab will buy it for you.
      GL

    • #69410
      javaid
      Participant

      actually it gets smeared only during restriction digestion coz when i run uncut sample it gives good bands but gets smeared after restriction digestion n even if i dont add the enzyme n incubate only with buffer… so i think it is nucleases which get active in buffer… now i need to purify my sample of those nucleases.. plz devise any method….

    • #69415
      SororSaudade
      Participant

      does the restriction enzyme you’re using have star activity? It could be cutting where it shouldn’t.

    • #69442
      javaid
      Participant

      no i does not have star activity whereas my earlier preprations of plasmids give good results after using these enzymes…

    • #71317
      Fanch
      Participant

      Hi all !
      I just "UP" this topic because I’ve the same problem..

      So, I want to digest my plasmid with Pst1 and Sal1 ( I use NE Buffer 3)
      After digestion my plasmid gets smeared, whereas my uncut plasmid gets the 3 "normal" bands.
      I thank the problem could be a star activity, so I tried with less enzyme according to NE book. Moreover I tried with just one enzyme and plasmid gets smeared.
      I thank that it could be a problem of nuclease contamination, because when I incubate my plasmid with just the buffer it gets smeared. So I tried with an other buffer, new water, and BSA. Same problem ! 👿
      Finally, I thank that it could be my plasmid prep ( I use a Qiagen miniprep kit). So I tried with another plasmid from a Midi prep. Same problem.. 👿
      Moreover, another co-worker in my Lab have the same problem and we don’t use the same electrophoresis or pipette or tips…
      But we use the same ladder, so I will try with a new ladder.
      It’s really strange ! 😯
      Thanks a lot for your help ! … and sorry for my english (I’m french)

      Fanch

    • #71319
      canalon
      Participant

      I do not think it can be your ladder, Fanch, because it should never be in contact with your digestion.
      But your results are weird, but did you check if your 2 tubes of buffer belong to the same lot? The problem might not come from you…

    • #71322
      Fanch
      Participant

      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?

    • #71336
      javaid
      Participant

      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

    • #71350
      canalon
      Participant

      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

    • #72789
      wangc
      Participant

      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!!!!!!!!!

    • #72790
      wangc
      Participant

      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.

    • #72833
      wangc
      Participant

      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.

    • #93388
      sinadas
      Participant

      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.

    • #93406
      JackBean
      Participant

      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.

    • #96828
      Eddie
      Participant

      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

    • #96853
      JackBean
      Participant

      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?

    • #101406
      kimsmarkin
      Participant

      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.

    • #103415
      jasen
      Participant

      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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