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confusion in real time PCR primers


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3 replies to this topic

#1 smilealie

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Posted 27 October 2010 - 06:03 PM

hello all,

i am new to this REAL TIME PCR.i need to work on a gene. i was going through the papers for primers each primer sequence differ from other in different papers for the same gene.I dont understand why this difference for the same gene and i dnt know which one i need to select?
i need to do with EVA green not probe based. kindly help me in this.
i hope you people will help me on this.....

thank you

alie.

#2 Abiotic_Kabir

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Posted 02 December 2010 - 06:49 AM

may be they used different software for designing primers. There could be several pairs of primers of a gene.
Use primer3 software and have a try!

#3 seed

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Posted 07 December 2010 - 03:32 PM

I also have a problem with primer and promoter sequences. What is the best software that I can use to find the promoter and primer sequences in Arabidopsis and tomato and how can I choose the best sequences? I have tried using primer3, however, I didn't get the same primer sequence as in previous journal and same goes to the promoter sequence.

#4 WSN

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Posted 10 December 2010 - 04:41 PM

Different from full length gene amp, real time PCR means only to quantify a gene sequence, so it doesn't matter whether you look at gene's head, body or toe, you may have people counting heads, while others counting toes, they for sure will use different pairs of primers. The primer design software will consider the penalty from having some homology between pair of primers or to other gene sequence or cause primer secondary structures that affect PCR efficiency, it will give you a list of suggestions with scores reflecting the quality according to software's algorithm. it's up to authors which one to pick.




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