Adding Ns cannot work as computers will search for exactly the sequence that you submitted/ Use either Primer3 or primer blast software searches and blast the primers individually, You will get a genomic position for each primer and just subtract the smallest number fromthe largest number for the finds that are on the right chromosome to get the amplicon stze

In addition to Paul Rutland's suggestion, you can determine the amplicon size as follows: Copy your FASTA sequence from NCBI and paste it into a word. Next, locate your forward and reverse primers (remember to convert the reverse primer into its reverse complementary sequence). In the search box, identify the target region between the primers. Once you've done that, count the words within the target region (you can find the word count by going to the "Review" tab in Word).

It's good to search literature:Article Molecular detection of vancomycin resistance and methicillin...

Plus if you know your target you can use primer blast to find your primers in the target sequence. Just use it with specified primers.

I find MUSCLE also works well for aligning primers.

Muhammad Ishaq Paul Rutland

I follow the suggestion that Paul provided, but when I copy the FASTA sequence, I can't find the forward primer in this sequence. I've tried several FASTA sequences, and none of them have been successful.

what species are you working with Thalita Camilo da Silva ?

It's for bacterial vanB? I was having difficulties finding the forward primer. In one article they say 463, others say 529bp so that doesn't help much either. I think it's the 529bp, I found both primers in KT003981.1 Enterococcus faecium strain 09_1818 VanY, VanW, VanH, VanB, and VanX genes, complete cds but with a mismatch. The problem was a mismatch in the forward primer (1 base: try a search for GCTCCGCAGCTTGCATGGA). Hope this helps...

In the paper attached by Dominika Kadlečková the primers are listed as

vanB 5′ -GCTCCGCAGCCTGCATGGA-3′ 463 [34]

vanB-R2 5′ -ACGATGCCGCCATCCTCCT-3′

Forward: GCTCCGCAGCCTGCATGGA

Reverse: ACGATGCCGCCATCCTCCT

and these primers match exactly yout sequences and give a size of 463. Domunika will probably have a better explanation of amplimer sizes than I can give but the size effect may be species related

I came late here, though this is my suggestion here; I'd advise you try running an Insilico pcr here...... https://genome.ucsc.edu/index.html. This has always worked for me, and I have recommended it to several researchers. I hope it works out.

Joseph Japhet Paul Rutland Dominika Kadlečková Muhammad Ishaq

I greatly appreciate the responses and tips. For this primer, I was able to find it through the hint in the article. However, I'm still unsure about what to do when I can't find it in the article, for example, for this primer pair:

• Forward Primer: blaKPC_FWD TTGTTGATTGGCTAAAGGG
• Reverse Primer: blaKPC_REV AACCTGCGGAGTGTATGG

I found the reverse sequence in this article ("Rapid low-cost detection of Klebsiella pneumoniae carbapenemase genes by internally controlled real-time PCR"), but I couldn't find the forward sequence in the article. When I input it into BLAST, I also can't find it. How can I resolve this issue?

Thalita Camilo da Silva Try alignment of your primers and target sequence. It should align even with mismatches or other problems. Then just paste it somewhere and count what is the length. You can try https://www.ebi.ac.uk/Tools/msa/clustalo/ and sequence of OP784923.1 (I blasted the primers first with a search of only the target organism K. pneumoniae. I Found hit which was in both blasts and you can use like your sequence if you don't have your own). Hope this helps.

hello, I dont know any thing about this matter. sorry