Erroneous agarose gel bands - (Nov/22/2004 )

Last week, I did a practical class where we used PCR to amplify a gene and ran it on a 2% agarose gel. 2 bands were present, one at approx. 1000 bp and one at 800 bp.

I'm now tackling the questions set by the demonstrator and am perplexed by this one:

"You have to repeat last weeks PCR reactions but now all reactions give you a band at the same position (c. 200bp) when you run them out on the agarose gel. What has gone wrong, and what will your first steps to try and correct it?"

I had initially thought that perhaps in this situation the gel may have been left for too long and the smaller band had run off the gel entirely and the larger one had migrated too far, but if this was the case, the ladder would have also run too much accordingly so it would not be identifiable as being 200bp long?

Does anyone have any ideas? I'm not posting this to 'cheat' on my work, it's that thinking about it and getting nowhere is driving me mad!

Hi

I am guessing that the other bands (1000, 800bp) are still there in the hypothetical situation, in which case you probably have a contaminant of some sort in the PCR reaction. For this reason always run a negative control (-ve control) )with your PCR (a reaction with no template. The template is usually replaced with water to make up the reaction volume), if you get bands in your -ve then you have a contaminant.

Solutions: Test your reagents for contamination (throw out your water immediately, it is the cheap one) by substituting reagents one at a time with new ones, and re-PCR running only a positive and -ve reaction, to save on ingredients.

If this isn't the solution, then try going to the library and looking for books on PCR, there should be a fair number. I suspect that the questions are meant to head you to the library.

Good luck

Hmm it could also be that the 200 bp reaction is the wrong one. Can you be more specific what you amplfied and from what template?
Were you supposed to get two bands in the first reaction?
In other words did they tell you, how large the amplificate should be, according to the primers used?