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" Exon/exon junction cannot be found for submitted PCR template. "--how to bypass, or work with?

Hi everyone, I'm a lowly undergrad doing research underneath a Professor. Could someone help me out and tell me where I am going wrong? The professor told me she would always reply in a timely manner but this can mean anywhere from an hour to a day, and time is money friends!

I went to genome browser and looked specifically for the Mouse Gene TNFSF10, and copied the refseq into word. I then found the beginning primer at 1 to 263 nucleotides. I found the next exon, the reverse primer, at 8905-9043. So, I took the refseq into ncbi's primer blast tool. The settings of importance I think I should mention are: 2GC clamp (I require either 2 or 3 of these in primers) and primer must span exon-exon junction.

I'm also looking for primers with no more than 200 nucleotides in length, so unless if I misunderstood something which is very likely, how do I make it so my reverse primer isn't so big?

Thank you, and sorry for this mess.

exon gene primer splicing intron
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written by lsizemor • lsizemor

Try this option.

Go to the primer design tool that you have found. Put RefSeq (NM_009425) accession for TNFSF10 in accession box. For the initial try just hit Get Primers button after selecting "show results in a new window" option. Once you take a look at the results (that may take 5 mins or so) go back and change any parameters you want to experiment with.

I see a couple of primer pairs in the default results that span exons but depends on what you are looking for.

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written by GenoMax GenoMax

Hi there, I did it like this but when I started messing with exon-exon junction spanning I got the same problem I was originally with.

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written by lsizemor • lsizemor

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