Dear all,
I want to do a non-denaturing DNA-PAGE and I didn't found any info regarding stacking and resolving gel for the purpose in the internet. Do they need, like in protein-PAGE, a stacking gel?
Thank you!
Hi there,
Yes stacking gel is also required in order to improve resolution of the separating gel.
Here is a calculator for preparing native gels: http://www.sciencegateway.org/protocols/cellbio/protein/sdspc2.htm
Hi Dominique
In the calculator you have referred does 1M Tris(pH 6.8),Tris (pH8.8) means Tris-glycine? If yes I do have a severe confussion. In most of the documents I have found they are making the resolving gel in 5X TBE buffer (pH8.3) and using 1XTBE as running buffer for detecting PCR products.So what should be the buffer pH for stacking gel??
Hi Debarati,
As in my experience, It is not necessary to use stacking gels for DNA PAGE. You can use continuous gel withTBE.
Hi again,
It's my mistake! I gave you recipe for protein native PAGE! Sorry about it... No need of stacking gel for DNA.
Just to clarify. The stacking gel is the zone where the stacking process occurs although it also occurs to some extent in the sample well . The stacking gel itself does not cause the stacking. The stacking, that is the concentration of all the analytes in the sample into a very small volume at the beginning of the separation is dependent on the buffer system used.
The commonly used Laemmmli system is a discontinuous buffer system (the buffers in the electrode and separation gels are different) : There is a Tris-glycine buffer in the electrode compartments and TrisHCl buffer in the separation gel. The initial analyte concentration is caused by the trapping of the analytes, in a small volume, between the potentially fast moving chloride ions and the potentially much slower moving glycine ions. The stacking of the analytes, in order of their respective electrophoretic mobilities, occurs at the sharp interface between the Cl- and the glycine. This process can occur in the sample well even without a stacking gel. How much an improvement the stacking gel makes may sample dependent. The separation between the analytes becomes observable after they have moved into the separation gel and are detected there.
In the case of a continuous buffer system some stacking may occur when molecules in the sample reach the gel and the leading molecules are slowed by the presence of the gel matrix while those molecules that are still in the sample buffer are moving more rapidly and so, to some extent, catch up with the leading molecules ; in effect concentrating the sample. These effects can be detected by observing marker dyes or coloured proteins.
Thank you all for your suggestions,I will do likewise and will let you know.
There is no need for a stacking gel for DNA electrophoresis on polyacrylamide. You can make the gel with TAE or TBE. The acrylamide will need to be at least 4% to obtain good separation and for resolving single base differences in size it is better to use 8-10% denaturing (i.e. with urea) gels.
Stackers are only necessary for protein gels.
I ran the gel at 15 mAmp,100V for 75 mins.The gel ran fine.Then I tried to change the current to 10 mAmp but the voltage automatically changed to 300V. I tried to lower the volt keeping the mAmp constant but the machine showed error message (E1).
So I was compelled to run the gel after 75 mins at 15 mAmp 300V for another one and half hour.Results came but at the middle of the gel a bow was created. Due to high current? I was using BIo-RAD Mini-Protean Tetra cell apparatus to run a single gel containing PCR products and loading dye with 1X TBE.
What is your purpose to use a PAGE gel (instead of regular agarose gel) to run your DNA? What is your DNA sizes (from your attached picture)?
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