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finding the corrosponding primers


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#1 sadaru

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Posted 23 September 2013 - 07:09 PM

Dear all

 

I am very new to real time PCR. I need to quantify some citokynes  by real time PCR with sybr green for mouse cytokines. (the genes are IL-1b, IL-6, IL-8, TNF alpa and COX-2.

Since it's plenty of primers out there, the choice is not easy. Even I found some publications regarding that I couldn't find the relevant bp values.( For real time PCR ,I heard the bp should be low and the maximum is below 400bp)
Someone has any  experience to tell me with any published primer for these cytokines.
 

Thanks for any suggestion.



#2 Tabaluga

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Posted 27 September 2013 - 12:56 PM

I don't quite understand the problem. What do you mean that you couldnt find the relevant bp values in publications ? Published qPCR primers should actually give relatively short fragments as you said. If you meant that the size of the fragment is not mentioned in the papers, just paste the primer sequences into PrimerBLAST and you'll see where they bind and what size the fragment is.
As a general advice for primer design, its a good idea to use published primers but doesn't necessarily mean it will work for you just because they are published (i once had some published primers that didn't work for me). I usually order both published and self-designed primer pairs and I always try them in a gradient pcr first.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 sadaru

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Posted 06 October 2013 - 03:04 AM

Thank you very much. I think this is very helpful. Actually I knew about primer blast but i didnt knw how to use it. Now I know how to use. Thanks again for ur personal experience . I believe this information is quiet useful for my problem. smile.png






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