Smith: One should expect to see ladders each separated by 1 nt in such cases.
Mohammed: My suggestions: 1) running at 70V seems low. I do not see a reason for not using higher voltages. 200Vx45min should be OK. IT is better to not let the BPB front run out of the gel - so that you know whether the DNA had all been chopped into single nts. (how do you visualize the bands? Is the oligo end-labelled? EtBr should not work well for small fragments. But UV shadowing may work.) 2) boil the sample before loading (6-8 M urea should always be included in sample buffers). This is important because the enzyme might bind the DNA and slowly release them causing the smearing. 3) How does the uncleaved DNA alone (no enzyme at all) look on the same gel? If it is a sharp single band then you might need to pay extra attention to denaturing the samples before loading. If it is also smeary then it might be the gel system needs optimizing. 4) use 0.75mm gel. Thinner gels give better resolution. 5) I found that DNA ran just fine in the discontinuous tris-glycine system(Laemmli). In other words, the PAGE system routinely used for proteins (without SDS) works for DNA too, with the benefit of sharpening the bands. You may also consider experimenting with gel percentages(25%-30 % for example) so that you can see even single nts. The acry:bis ratio is not that important as long as you stick to one. Zhijie > On Sep 30, 2017, at 7:54 AM, Smith Liu <smith_liu...@163.com> wrote: > > your enzyme cannot give definite fragment. thus smear > > > 发自网易邮箱大师 > > 在2017年09月30日 19:28，Mohammad Khan 写道： > Dear all, > > I am working with an exonuclease and I run the digested DNA on a > 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system > and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a > 15 well comb. I run my gels at 70 V for as long as 4 hours till my undigested > DNA reaches half the gel distance. I use 20-30 nt long susbtrates. > > I am mostly not able to get distinct bands of the digested products but > rather get a smear. Is there any way to make sure that I get distinct > digested products rather than a smear? > > I am looking forward for suggestions from all! > > Thank you. > > Ciao!
