DNA Gel Extraction Problem - multiple bandings in gel extraction product (Sep/16/2005 )
I've recently encountered recurring problems with my DNA gel extraction. I would run a 1% agarose gel, then excise the specific band and do the gel extraction using a kit. But when I check the results I get multiple bandings!
Can someone help me on this? Could it be because of overloading the gel?
Thanks a lot.
There are many reasons for the problem. Upload your gel picture.
1. DNA extraction from agarose gel does not guarentee 100% purification of the desired-sized DNA. Overloading causes this problem, too. If you got the same bands to the original ones, and the not-desired bands got weaker, i would say, it happens. Just re-purify the desired band. Or increase or decrease the agarose percentage to adjust the condition for easy separation.
2. Did you check the buffers from the kit, including your water?
You may denatured your DNA - this can give multiple bands.
DNA sequencing software
>>I had same problem. When I decreased quantity of DNA in my sample, it became better.
>> In some cases decreasing % of agarose downto ~0.8 solve this problem
Firs of all, What is the size of your DNA? This is so important, If the size of DNA bigger than 6kb, you must use 0.8-0.7% agarose gel. I think that your DNA degraded. At this point, you must run the DNA slowly and your purification kit is also important. I use the Applichem DNA isolation Kit and you work well.
Thank you so much for all your input. Sorry I forgot to mention that the multiple bandings I got were actually multiply larger than my desired band, and yes, the bands were weaker than the desired band (but still a nuisance nonetheless!). So, I doubt that the problem is degradation of DNA.
Anyway, I tried to use 1.2% agarose, made sure I didn't overload this time and ran the gel longer (40 min at 120V) to achieve better separation and get a tighter band and avoid overlapping fragments, and hey, this time it worked! I'm so happy...
God bless you.