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Primer design HELP!

primers Eschrichia coli bacteriophage

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#1 MushyBrain

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Posted 18 January 2017 - 01:25 PM

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!



#2 bob1

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Posted 18 January 2017 - 07:08 PM

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



#3 MushyBrain

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Posted 19 January 2017 - 09:04 AM

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



#4 labtastic

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Posted 19 January 2017 - 03:09 PM

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.


Edited by labtastic, 19 January 2017 - 03:10 PM.


#5 MushyBrain

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Posted 23 January 2017 - 08:52 AM

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.


Edited by MushyBrain, 23 January 2017 - 08:52 AM.






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