I am amplifying a DNA sequence using thermal cycler PCR. The gene size is supposed to be ~1600, but I am also getting a strong band at 200-300 bp when running the PCR product. I have tired increasing the annealing temperature. The template I am using is a gene that was ordered. I was wondering what is causing this, and how to resolve it?
is the band present prior to amplification? run a few microliters to see if it is a degradation product in what you ordered. you say you ordered a gene - is this cDNA, or IVT RNA? either way aliquot this reagent to avoid freeze/thaw cycles and degradation that could lead to these bands
blast your primer sequences against your organism and see if there are other binding sites.
you can also decrease salts in the buffer for more specific binding.
I would first suggest checking the primers you have designed for amplifying this gene. you can check the specificity using insilico pcr followed by blast. if your primers are specific to your target gene then you should vary your pcr reagents in different dilutions.
How much template dna are you amplifying. Best pcr amplification uses about 30000 copies of template dna and it is very easy to start with too many copies when the template size is so small. This may be an over amplification effect. You could also try to increase the specificity of the primers by amplifying in the presence pf DMSO at any concentration up to 10%. Try running a dmso gradient.
I agree with Paul. This may be excess of template DNA.
Try lower concentrations of template.
- Badges
- Science method