I am doing the gel electrophoresis to check my DNA samples, and I found that after adding loading buffer and start running, the distance between bromophenol blue bands and xylene cyanol FF bands become much closer than before, and I repeat experiment several times, it still happen, and it seems leads to large fragment can be separated but small fragment can not. I try to change the gel concentration and running buffer, but still can not work. I have try 6% and 10% native PAGE gel, with 100V / 200V in 30 mins.
And the TBE buffer was prepared by ourself.
I can not figure why it happen, and I am not sure is there any step I make mistake.
Thank you.
Check your BIS-Acrylamide concentration? It is the only thing left, is it old? Bis-Acrylamide cross links the acrylamide chains and thus makes the pores rigid and hence better for small molecule separation. If the Bis-Acrlamide is not working you will not get the cross-linking you normally have. You usually only vary the Acrylamide concentration, and this variable alone determines the MW range of separation. Varying Bis-Acrylamide concentration is not recommended. However, its effective concentration can inadvertently be changed: it does age.
Some of things which can increase the resolution of your DNA bands include:
a) running the gel at a lower voltage for a longer period of time;
b) using a wider/thinner gel comb;
c) loading less DNA into the well.
d) Use of fresh buffers, APS and TEMED.
e) Make sure that the DNA ladder and sample deposits at the bottom of the well while loading.
f) percentage of gel will depend on the base pair resolution you need. So prepare your % gel accordingly.
https://www.nationaldiagnostics.com/electrophoresis/article/native-page-dna
Hi Wei
I hope you will find the solution from the above mentioned suggestions. You can also try 1.6% to 2% agarose gel and run it with 65V for longer time. You will find a better separation. Wishing you best.
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