One specific DNA band became 2 bands after gel extraction? - (Jul/09/2006 )
dear friends
I have purified The specific band which was 650 bp by gel extraction. after the purification I ran 3 microliter of it on 1.5 % agarose gel and I saw 2 bands one is 650bp( the sharper one) and the other is about 1300bp(very weak) ,accourding to company's instruction I incubated the purified sample in 95'C for 5 min and then put the sample in room temprature for 10 min and then ran them again and now I see 2 bands one is 650bp (the weaker one) and the other is about 300 bp (the sharper one ) can any one help me to explain what could have happend to my sample?and why I get two band after purification which are very different in size?
thanks
When you heated your purified sample at 95ºC for 5 min, was the DNA in water? If the pH of your water is off, you can get denaturation of the DNA (heating would expedite this process) and your single stranded DNA would migrate at around 1/2 of the dsDNA size. Sometimes you have to elute in water if that's what the company supplies or says is the only way to elute. If that is the case, add Tris-HCl (pH 8.0) to 5 or 10 mM to your eluted DNA to help protect it from denaturation. When you dilute your DNA and loading dye, use TE or 10 mM Tris-HCl (pH 8) to bring up the volume instead of water. We avoid having DNA in an unbuffered solution whenever possible, even in the short time of mixing it with dye to run on a gel.
Why would you heat your purified sample before loading it on the gel? What protocol/kit are you using?
As for the 1300 bp band, did you purify your 650 bp band away from one this size? If you didn't separate the 2 bands enough (or ran the gel too fast), you could get cross contamination.
Hi,
I'm guessing that you're doing restriction enzyme cut and then isolate and purify your DNA via agarose gel electrophoresis?
Could it be possible that the cohesive ends of two 650 bp fragments interacted with one another forming the 1300 bp thru hydrogen bonding?
I love MSGs! is right.
no I am not doing digestion what I cut from agarose gel was my PCR product which had multiple bands and I waited too long when my gel was running until my loading buffer was getting out of the cast and then I cut the 650 bp band so It cant be other contamination I dont know if DNA can attach to itself and make 3mer tetramer or something like that?
Why would you heat your purified sample before loading it on the gel? What protocol/kit are you using?
As for the 1300 bp band, did you purify your 650 bp band away from one this size? If you didn't separate the 2 bands enough (or ran the gel too fast), you could get cross contamination.
dear friend
when I heated my sample the DNA was in tris HCL buffer pH=8 10mM and when I m dilutting the DNA I am usind TE buffer .
Again, why are you heating your purified DNA? You are inviting denaturation. It's difficult to predict how partially denatured DNA will reanneal in just 10 min at room temp, and you might get different species on your gel every time you heat the sample and run a gel. Try running some of the purified product out without heating it on the same gel as the heated sample. The only common time when DNA is heated before loading on a nondenaturing agarose gel is the Lambda HindIII ladder, since long repeats on two of the bands will make them partially anneal together.
If you see more than one band again, and there was no ~1300 bp band in your original PCR that might contaminate your 650 bp, I'd try another purification kit (Qiagen or GeneClean work well). Believe me, if you purified your product on a minigel and/or ran the gel too fast, no matter how far apart the bands look on your UV box, there will definitely be a small amount of the DNA from the neighboring bands in the band you want. I'm not sure that's the problem, though, since you don't give specific information about the kit, sizes of the other bands, etc.