I eluted my amplicon from gel and checked its concentration after elution (column based kit) on 0.8% agarose gel. In the eluted product I found another band above my amplicon, which is very unusual as although it had some primer dimers, but no bands above my PCR product and also, I carefully cut out the band of my desired size only. Strangely, it appeared only after elution. What could it be? Although the band is very faint in the gel, will it be safe to use eluted product for downstream processes?
How many times did that happen? It might be just some accidential salty spill over from a kit solution in your prep.
a similar logic as Pranjal's answer....what if your amplimer contained a polymorphism .Then some of the double strand would be a normal/mutant heteroduplex which would form a bubble of single strand and might run slower (looks like larger) even though it is the same size in terms of base length. The n-n and m-m would both run at the same smaller length
yes that is a problem for the heteroduplex idea Tias..you are right.Do you have a picture of the 2 bands on a gel .It would be interesting to see how much bigger this band is and if it only appears in tracks with a sample in them
if you were thinking that this other band is an artifact from the kit purification process you could try purifying a water sample and see if the odd band appears .
As the band has appeared at the same position more than once it possibly cannot be an artifact, and also it is not possible that you may have cut them out together because they are quite distant from each other. In this case you may again elute out the desired band to separate it from this higher molecular weight band, and then check on gel again whether this band reappears or not. If it reappears than probably this band is assuming some structural variation as mentioned in earlier answers. And if you get single band this time then the problem is solved. Good luck !
Super. Now can we have some labelling or explanation for the top, middle and bottom, pictures?
Actually, it doesn't help!
Its strange that the two PCR products appear to be of different sizes - is that possible?
So here's my wild guess, based on the lack of information that you have given us: you started with a circular plasmid - probably quite large amounts (as that's what most people on this forum seem to use, although it isn't necessary). You checked a small amount of that PCR for your product, then ran a large amount and purified everything, The circular plasmid probably ran with the PCR product. During the purification, the circular plasmid became linearised and now migrates as the larger band. So for this to be right, that "new" band must be the same size as the starting plasmid (linearised). And if you cut it out of the gel and try amplification, you will again get a product of the right size.
Dear Margison,
1. True, I have used a circular plasmid as my PCR template and have used less than 100 ng of it, which I assume is not an unacceptably large quantity. I really don't know what large quantities most people in this forum use.
Anyways, I got some help from another forum. People facing similar problem may find it useful.
http://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/
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