dimers of dna (not primers) following digest or pcr? - (Apr/25/2006 )

Hi,

I have been doing RE digests and also PCR on a vector (pcDNA3.1) containing my desired fragment (840bp) to try ligating and expressing for functional studies. Multiple times, I have observed an extra band in my gel which is approximately double the size of my desired fragment.

Has anyone had this occur before, and do you know what it is?

I have even had the circumstance where I have obtained a PCR product with only the desired band, done a PCR on that and then obtained the double bands like in the attached picture.

Any assistance would be greatly appreciated

wow, I suspect you are using way too much template in your PCR reaction. You have a great deal of smearing in that gel, and all that stuff that is remaining in the wells isn't a good thing either.

I would not trust a band that I had purified from that gel; all the smearing suggests a bunch of non-specific product.

Others may have different ideas, but if I were you I would try these things: clean up your gDNA a little and try much less in the reaction. I would also monkey around a bit with the annealing temp...try a gradient if possible.

good luck

hi
i agree with aimikins and would not trust such a gel. But i try to avoid pcr on pcr... That allows a non specific fragments amplification... far better to do 3pcr on a plasmid than 1PCR on a pcr product...

having too much DNA template in the reaction enhance possilities of smears and non specificity

Thanks for that advice both of you, I am surprised that noone in my lab told me the same thing...

I will trying to PCR with a few different amounts of template to get the best result.

Thanks

Wow, I didn't realise that was from DNA being in the wells. All of the gels I have been running this year have looked like that.

Is this because I have been loading too much sample into the wells, or something to do with the way it is being run? I have been using a 1% agarose gel and running at 120 volts for around 15 - 30 minutes. I have been loading my whole PCR product (50 microlitres) and then purifying from the gel.

DO you have any recommendations on what I should try?

Thanks so much for your help everyone

Hi,

I have been doing RE digests and also PCR on a vector (pcDNA3.1) containing my desired fragment (840bp) to try ligating and expressing for functional studies. Multiple times, I have observed an extra band in my gel which is approximately double the size of my desired fragment.

Has anyone had this occur before, and do you know what it is?

I have even had the circumstance where I have obtained a PCR product with only the desired band, done a PCR on that and then obtained the double bands like in the attached picture.

Any assistance would be greatly appreciated

gstar, have you spec'd your DNA? I suspect your chromosomal preps are not so great...perhaps the DNA has some protein contaminants and that is why it is being hung up in the wells...at any rate, I think template quality and quantity are your culprits

your protocol for running the gels seems fine, that should not be the source of the problem you are running them a bit fast (unless you're using the sodium borate buffer system?), but I do not think that is the entire problem for certain

Hi,

After looking back at my previous gels I have realised that even the lanes that are empty have glowing wells. COuld this be caused by the fact that in our lab we don't destain? It could be that some of the stain has run into the wells before I photograph?

hi
i don't destain the gels that contain EtBr when pouring. and i don't have remaining DNA in wells.
That may occurs by an overload or too much salts in your sample.
what do you mean by stain your gel? you mean that the agarose gel does not contain EtBr before being poured? you may try using EtBr containing gels.