Frequently, I've got an error on Primer-Blast that says: "Exception error: CFastaReader: Input doesn't start with a defline or comment around line 1. To solve it, I tried different browsers, but it didn't work. To figure it out, I used my pre-checked primer, and shockingly, the error was repeated.
It is not the browser problem. Either you are using file upload option with file having errors, or something not right with the chosen options.
That error means there is a formatting issue with the input Fasta file.
For the PRIMER-BLAST in NCBI you have to give the FASTA format of the PCR template and the sequences of both the primers in 5'-3' direction.
I think there is an issue with your input files, check them properly.
Or else let me know specifically what is the purpose of checking of primer.Do you want to identify if your primer will work properly, I mean if there is any chance to amplify other genes along with your target gene? Then you can simply go for a nucleotide-BLAST (nBLAST) with your primer sequence, from the provided list in the result you can easily find out the genes it will amplify. Do the same for both the primers. From the result you can calculate the length of the amplified product also. I think it will help.
Most likely the problems occur during the uploading. To avoid this a copy and paste could help.
Use Microsoft Edge browser, do not use Chrome browser, after typing, wait for about 30 seconds, a new jump window will be added, and the search results will pop up.
Just try other browsers like Microsoft Edge. It was helpful for me.
Please choose left to right text direction. by setting the Primer direction from left to right, probably the problem will be solved.
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