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designing primers( selecting target sequence/amplicon design)

qPCR DNA PRIMERS

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3 replies to this topic

#1 ntsalihah

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Posted 06 January 2014 - 04:00 AM

Hi everyone,

 

Okay this sounds a bit dumb but all these are new to me, so please help in anyway you can.

 

I'm trying to design a new primers (let's say for a bacteria). I know for amplicons you're suppose to select 75-100bp, avoid secondary structures etc...

 

but how do you go about selecting the target sequence? Is there some kind of database where you can see the dna sequence of a bacteria and can select a target sequence/amplicons on that? and thus the primers.

 

I'm sorry if I'm not being clear...please any help would be much appreciated.


Edited by ntsalihah, 06 January 2014 - 04:02 AM.


#2 bob1

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Posted 06 January 2014 - 12:19 PM

This sounds like homework to me.......  so my answer won't be complete, but should be enough to get you started.

 

Have a look for genbank.

 

What type of PCR are you doing (for what purpose)?  qPCR (real time) or standard?



#3 ntsalihah

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Posted 07 January 2014 - 02:14 AM

It's not homework I assure you. I just never design primers before and only study in a classroom (and they only tell you how to design primers , not exactly how to do it in real life ) instead of practical experience. and practical experience with PCR only and with already prepared primers.

 

And It's for qPCR.

 

thnak you



#4 bob1

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Posted 07 January 2014 - 12:07 PM

There are databases of validated (published) qPCR primers out there, but I don't know the websites off hand.  Have a look at the Nucleic Acids Research database issue.

 

Check out PrimerBlast on the NCBI website.







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