I am planning to prepare a 100-1500bp DNA marker using endonuclease digestion of a special plasmid. I am wondering if the presence of sticky ends can affect gel migration of generated fragments. Agarose gel electroforesis is surely not enough precise technique to distinguish small differences in MW, but I am worried about possible bands' smearing, especially the smallest ones. Do you think this problem may occur?
I past half of my life preparing DNA ladder markers using lambda DNA cut with HindIII, or PstI, and phiX174 cut with HindIII. I used them separated or mixed together, depending on the fragments expected sizes and the plasmids used. I never found I single artifact. But the time (this is probably the most important issue) to prepare the ladders and check they are fine, the investment in restriction enzymes and DNAs is not worth it compared with the amount of different DNA ladders available in different suppliers, as well as their competitive prices. Most of the time, we use in the lab half the recommended amounts with optimal results.
small DNA bands do get smeared occassionally in the gel electrophoresis. Smearing is also observed when your DNA sample have excess salt or if u have loaded too much of the sample. Degradation of the DNA or contamination of DNA with proteins also cause smearing effect
The ends will not cause problems. Degradation, overloading or too much salt in the sample will.
I think that the problem is due to protein contaminations of your samples, however, if you think that the problem is due to the protruding ends, why not try to fill the protruding ends with kleenow fragment of DNA-Polymerase.
Commercial DNA markers are often prepared by routine restriction digests and have sticky ends. So don't worry about it at all.
I past half of my life preparing DNA ladder markers using lambda DNA cut with HindIII, or PstI, and phiX174 cut with HindIII. I used them separated or mixed together, depending on the fragments expected sizes and the plasmids used. I never found I single artifact. But the time (this is probably the most important issue) to prepare the ladders and check they are fine, the investment in restriction enzymes and DNAs is not worth it compared with the amount of different DNA ladders available in different suppliers, as well as their competitive prices. Most of the time, we use in the lab half the recommended amounts with optimal results.
I agree with Gonzalo Martínez-Rodríguez, I had the same, preparing our own DNA ladder markers.
I do not prepare my own DNA-ladders but i often digest plasmids resulting in sticky or blunt ends , respectively. According to my experience it is irrelevat for the passing distance on agarose gels which ends are present- even if you have smal fragments around 100 bp.
Generation of DNA ladder will be a project of a our student organisation. It will be performed by younger students to improve their skills and gain some experience. And I am sure it will come in handy in laboratory sometimes. Thank you for all of your answers. You helped a lot.
Dear Olga Lihoradova,
I appreciate your advice but I do not think I really need it. I would say I understand this technique and its physical basis quite well.
Best Reards
Dear Olga,
Of course I know the resolution of agarose electrophoresis. It is low and the difference in lenght of few bp is rather not possible to observe. I was thinking of different problem. After digestion e.g. with BamHI we obtain 4bp long overhangs on the both ends of 100bp DNA fragment. In high concentration of DNA we may observe hybrydisation of sticky ends and formation of metastable dimers/ oligomers. This phenomenon could lead migration delay and in effect to smearing and non-sharp bands. Similar effect is obserwed in EMSA technique, but of course affinity of protein towards DNA in EMSA is higher. So this was my way of thinking. And I am sorry about this misunderstanding. I should have described it clearer.
The overhangs may influence DNA mobility in polyacrylamide gels especially when fragments are below 300 bp. Generally we see retardation and smearing which may be caused by intermolecule interactions as already mentioned by Dawid. The anomaly is particularly significant after the BamHI digests which leave 4-bp overhangs. After filling the sticky ends with dNTPs and a polymerase treatment fragments migrate normally. Migration of DNA in agarose gels is usually unaffected by the any of the overhangs.
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