PAGE is known for its higher resolution of the bands of low molecular sizes than agarose gel.

But your amplification as well as the digested products should run fine in agarose gel also and should be discerned by the Uv visualization.

One thing to do is to use enhancers like DMSO or Bovine Albumin during PCR or also increase the number of cycles like use 40repeats and you can also use PAGE for the resolution of the digested product

PAGE is known for its higher resolution of the bands of low molecular sizes than agarose gel.

But your amplification as well as the digested products should run fine in agarose gel also and should be discerned by the Uv visualization.

One thing to do is to use enhancers like DMSO or Bovine Albumin during PCR or also increase the number of cycles like use 40repeats and you can also use PAGE for the resolution of the digested product

polyacrylamide would be preferable. You should see from 10ng to 200ng of your products. Plus, you can always be sure is the size of fragments is good, if you use low-range dna marker.

Polyacrylamide gels are superbe for the sizes of DNA fragments between 750-25bp. Above 750bp it will be rather difficult to resolve the fragments due to staking and you may have to prepare gels of 3% which becomes very tedious.

PAGEisjust great for resoultion and with practice and adjustments to the amounts of ingredients and sample size its the only way to go.

Thanks for the advice. I really like the idea of the bioanalyzer because my problem is the low amount of DNA present at the 100bp and 200bp fragment sizes.

In my research I have fragments of digestion with 146pb. In agarose gel I don´t see then. In PAGE I see but not in a very high quality because the fragments present a low amount of DNA. PAGE can show differences in samples with same number of fragments with same size, this occur because polymorphisms in the nucleotides between different samples can allow the fragments run different in the gel. Be careful with that and PAGE will be the best for you in my opinion.

It's not so much which kind of gel, rather, the staining method matters most. Think of ethidium bromide, silver staining, SYBR ... Moreover, any stain binds to the DNA molecules in a quantitative way, i.e. shorter DNA fragments bind less stain, therefore are less intense on the gel. In a digest, you start with a certain amount of one larger molecule (binding more DNA = more intense band) and end up with the same amount of molecules (two or more) but they are shorter molecules (binding less DNA = fainter bands).

@Amy L

You have put the question nicely....

1.PAGE is used to see the if there is less separation/(less bp) distance/ for higher resulation....

2.In your case there is 100 bp difference 3% Agarose gel is enough to differentiate it...no need to go for PAGE.

3. If it is related to faint band check with PCR programme/components...Load your uncut sample side by the digested well as a control.....

4. If possible put gel it is better to explain you....

good luck....

6-8 % pages (acryl-bis 19:1) are able to discriminate fragments 10-300 bp with small differences (5-10bp) . Gels must be stained after the run. The detectable DNA quantity depends on the dye used. If the well is tight you can detect about 5 ng staining by Br-Etidium. Can you start the experiment with proportional higher DNA quantity?

You must distinguish 2 parametres with any type of electrophoresis: 1/ resolution & 2/ signal intensity

Denaturing polyacrylamide gels are used because they can resolve small differences in length for small/medium fragments, ie they resolve small length differences to the base pair. On the other hand, agarose gels, even at high concentrations separate fragments, but not with high resolution.

Signal strength is dependent on the dye and the target quantity. Common non-specific dyes such as EtBr, GelRed, SYBR Green, etc. bind to the minor groove of the DNA. Signal intensity is directly proportional to the molecule length and its abundance.

Your problem is due to low abundance of material. This issue is not solved by changing the type of electrophoretic matrix. Diffusion might attenuate your signal, but for 100bp fragment sizes in high concentration agarose gels (1,2%–4%), it should not be an issue. Besides, your not trying to distinguish between two products of very similar size, but trying to detect the presence of a small fragment.

Using a different type of staining method (ie silver staining) could be a more direct approach.

Radiolabelling your digested product or a Southern blot could also be a technical alternative.

Good luck

Okay, I am beginnning to understand your problem a bit better, by and by :-)

One thing we do with agarose gels and restriction digests: we incorporate ethidium bromide in the gels, so we can take the gel to the UV box early into the run (after say, 20 minutes of a total run time of approx. 1.5 hours) - then you will see small bands much sharper and better. These bands get more diffuse the longer the run, and in the end often 'disappear'. You can put the gel back into the chamber after such an early picture, and take another shot after a while, and one in the end of the run.

To run double strand DNAs you need 6-8% polyacrylamide gels in TBE or TAE not-denaturing (without urea). Denaturing polyacrylamide gels are performed to run single strand DNA , i.g to run DNA sequence reactions using the chain-termination method developed by Sanger we use to run denaturing 6-8% polyacrylamide gels containing 6M UREA

Detection of the PCR results in polyacrylamide gel is more sensitive than in agarose gel (more than 10 time).

I am thinking of using GelRed or Redsafe for PAGE and cut off silver staining steps:

+ add GelRed or Redsafe to casting solution and cast the polyacrylamide gel. 

+ Load non-denaturing PCR products on PAGE

+ After few hours runnnings, remove glasses from electrophoresis aparatus and visualize on UV-vis

Anyone tried it and recommendations?