I use confirmed homozygous mutant and homozygous normal samples for tetraARMS PCR optimization. everytime I run amplified products on gel I observe three bands instead of two bands. DNA samples and primers are not a problem these are verified at various times. The reaction was repeated with commercially available PCR ix and component but the results were same. What should be the possible reason of this and how to get best and accurate results?
Either it's an artifact or it's contamination.
What do your controls look like?
Katie A S Burnette observed a similar pattern. I tried using different DNA samples, made new working aliquots of primers, and even increased the length of the primers. I also ran a Primer-BLAST, but it didn't show any products other than the desired ones. I am clueless about the other band that shouldn't appear.
I agree with Katie A S Burnette . If you are getting a band when you should not then either there is contamination in which case the no dna control sample will amplify this band or the annealing temperature is so low that your mismatched primers are amplifying the wrong allele but no template controls will show any contamination
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases