Hi! I am working on the RFLP analysis of Infectious Laryngotracheitis Virus using thymidine kinase gene. The primers are prepared in accordance with the previous literature and their sequence is forward: 5’-GCTGGGCTAAATCATCCAAG-3’ ,and reverse :5’-GGAAGCGGAACATTACGAAC-3’. Even though the primers are aligning with the thymidine kinase gene, they are not showing any results on primer 3.
I know this might sound obvious, but did you put in the reverse complement for the reverse primer?
Could be your viral isolate is divergent sequence wise from others in the literature at one of the primer binding sites. If there is an SNP near the 3' end of the primer binding site that can disrupt amplification.
@ Katie A S Burnette I did use the reverse complement for my reverse primers. They are showing 100% alignment on BLAS T and MEGA as well. However, when I checked these primers through Primer 3, they did not produce a satisfactory result.
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