Primer design HELP! - (Jan/18/2017 )

Hello everyone,

I am struggling with some primer design and my training in molecular biology is limited. After designing a primer to select for Shiga toxin 1 for several strains and serotypes of E. coli, I noticed after blasting my primer sets that I was also matching Enterobacter phage and Stx 1 converting phage. What I am trying to determine is, if doing traditional PCR, will I end up selecting for both my target sequence in E. coli as well as where my primers anneal (100% match through blast) to the phage? There is a slight variation in amplicon size but perhaps this is something I really don't need to worry about?

Any info would be helpful

Thanks!

If the phage is present in your sample then you would get the phage DNA amplifying as well as the bacterial.

Okay, thank you, that was what I was thinking but no one I work with knew for sure

Can you design primers that anneal outside your Shiga toxin 1 gene in a region of DNA that is unique from the undesired phage DNA? 

If so, then you could specifically amplify the Shiga toxin 1 containing those extra flanking regions with those primers. Gel purify the PCR product. Use this as a template to amplify your desired Shiga toxin 1 region with nested primers.

On the other hand if you just amplify both pieces, and they are sufficiently different in size that you can separate on a gel, then you should be able to gel purify out the desired product and continue downstream work with that.

labtastic on Thu Jan 19 23:09:09 2017 said:

Can you design primers that anneal outside your Shiga toxin 1 gene in a region of DNA that is unique from the undesired phage DNA? 

 

If so, then you could specifically amplify the Shiga toxin 1 containing those extra flanking regions with those primers. Gel purify the PCR product. Use this as a template to amplify your desired Shiga toxin 1 region with nested primers.

 

On the other hand if you just amplify both pieces, and they are sufficiently different in size that you can separate on a gel, then you should be able to gel purify out the desired product and continue downstream work with that.

Thanks labtastic, I was thinking of doing something similar simply to distinguish phage from bacteria. This is only a detection experiment and samples are not being used again downstream. We are amplifying other segments of E. coli (O-antigen specific) so we know that it is what we are looking for but we may have a number of false positive Stx1 results if I can't get the phage out of our sample.