I am doing an enzyme activity assay of an endonuclease which uses annealed oligo DNA as substrate and cleaves at one strand and releases a single-stranded DNA fragment. This strand that is gonna get cleaved is Fam-tagged which can be visualized under Typhoon.
Idealy, the band of the substrate should be of the same size as the single strand marker (which is the exact strand that was annealed with the opposite strand and then served as the substrate of the reaction) and below it, the band of the cleaved shorter DNA fragment. My problem is that I got two bands other than the product, one is of the correct size as the marker, the other is larger, indicating that the denaturation is inefficient.
Previous experience is that incomplete annealing of the oligo DNA would cause the double bands of the substrate, while upon complete annealing, only one band would appear.
Details of the experiment
After the enzymic reaction, I added 20ul quench buffer (95:5 mixture(v/v) of formamide to EDTA) to the 10ul reaction mixture. Then, the samples were heated at 90℃ for 20 min and then immediately placed on ice. The Urea PAGE gel was prerun for 20 min. After sample loading, the gel was run at 200V constant and visualized under a Typhoon.
I read that the buffer could be heated to 50℃ before electrophoresis, and gel can be run at 300V to maintain the heat so that DNA can remain single-stranded. Can anyone give me more suggestions on how to achieve complete denaturation of the oligo DNA or better ways to conduct the experiment? Or is there any other explanations you can think of?
we usually use a loading buffer that consists of 95% formamide, and loading dyes. mix with your DNA sample 1:1, denature at 95C for 5 min and then load on your Urea-PAGE. The temperature will denature the dsDNA and the formamide keeps them denatured.
Thank you Mario Vieweger . I use the same loading buffer, though sometimes I double the volume of the loading buffer. However, I did find that sometimes the DNA were not fully denatured as there would be a band of bigger size that that of the single strand. May I ask on what conditions do you run your gel, for example the voltage and the buffer temperature? Many thanks!
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