Gel Electrophoresis Smearing Problems - Molecular biology always a mystery! (Apr/02/2011 )
This is my first ever post in this forum!
I have few problems that occured when I doing my mini molecular biology project. It was so stressful and mind-blowing to troubleshoot the problems and leads to difficulties for me to make a disscussion on my project.
I encountered heavy smearing problems after I did gel electrophoresis. I have repeated it several times but smearing still occurs. Electrophoresis was done on 1% agarose gel. cDNAs that I used are cDNAs of plant extracts (hibiscus rosa sinensis) treated-breast cancer cell lines.
The image can be seen at this link -> http://uploadpic.org/storage/2011/ZITLp8m3tVgNWBMpFcxIJiI2d.jpg
Notes that ACP reffered to annealing control primer.(Seegene's DEG kit)
So, what exactly the main causes of the smearing and what is the other factors that may involved in gel electrophoresis? I tried to perform further optimization but due to time limitation, I can't.
smearing is caused by overloading and by multiple species.
are you preparing the cdna from a specific mrna or are you making the cdna from all of the mrna?
Sometimes the smearing is caused by your DNA in the process of degradation. As in, it is cut up into all kinds of pieces by DNases. Some ways to fix it include eluting your DNA in T.E. instead of water, or using a new stock of T.E. in case contaminated, or keeping your DNA at colder temperatures.
@mdfenko : cDNA is actually prepared by conducting reverse trancription of the extracted RNA from the extracted cell lines..
@chimpsarehungry : thanks..i also thought that my smearing problems is largely due to degradation of DNA..
are you preparing cdna with a specific primer? if not then smearing may be more due to multiple species of cdna than to degradation.
if the smearing is below the band then it may be more due to degradation. if it is also above the band then it may be more due to overload and/or multiple species (your picture looks like smearing above and below bands).