PCR Bands

[dttom]

I am trying to amplify a portion of DNA from a genome (and get no band), I also included a positive control while I was doing a temperature gradient from 37c to 55c. For the (+)control, I got one band (with its size fit) at 55c but none at 37c. If I got no band with a higher temperature I can deduce it is due to the inability of primer to bind onto genome template, but now it is the lower temperature which showed no band. With a lower temperature, multiple bands due to multi-target makes sense, but I do not know how such a band pattern is result. Hope get any help, thanks.

[CharonY]

A simple explanation is that the temperature is so low that it binds at random positions spread out through the chromosome. If they are too far apart and/or the binding is random enough you will not amplify anything, either.

(37 is really low for a standard PCR).

Edited July 29, 2010 by CharonY

[dttom]

Ok, I see the reason.

By the way, as mentioned, I could not see any band even I conducted a gradient from 37c to 55c. To check if it is due to genomic DNA defect, I may include a positive control. But in the PCR mix (forward and reverse primers, dNTPs, DNA Pol (I use Taq), buffer, genomic DNA and water (please tell if I miss any ingradient)), another possible source of error might originate from the pair of primers, I would also like to know if there is any method that could ressolve the error source to one kind of primer (whether the problem is based on the forward or reverse primer).

[CharonY]

Getting no products is indeed often due to primers. You should check whether they are as "optimal" as possible (e.g. checking for repeat runs, unspecific binding, GC content etc.). While one could ascertain proper binding of individual primers to the template, most of the time it is easier and cheaper just to generate new primers. If the GC content is high you may want to use additives to lower melting temperature, for instance. I also assume that the buffer also includes the ions that the polymerase needs (usually Mg2+). If it is separate from the rest of the buffer you can also try changing that concentration.

[MedGen]

You can test your primers (providing they aren't degenerate) using an in silico PCR that's available on the UCSC Genome browser, also providing the organism you are amplifying from has had it's genome sequenced:

http://genome.ucsc.edu/cgi-bin/hgPcr?org=Human&db=hg19&hgsid=167311066

 

This may allow you to see the expected size of your product. I'd also say that when running a temperature gradient it's best to run the gradient from about 3 degrees below the Tm (use the Nearest Neighbour Tm if you can as this takes into account the presence of divalent cations, i.e. Mg2+) upto 72^oC (optimal Taq temperature).