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RT-PCR primer design

rt-pcr primer design

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#1 eikichi91

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Posted 30 June 2012 - 07:06 AM

Hi,

I want to ask about how to design primer for RT-PCR. Specifically, i want to design primer based on the human mRNA encoding the c-myc, p53, caspase-3 and bcl-2 gene. I have no clue where to start and what to do...

thank you..

#2 pcrman

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Posted 30 June 2012 - 07:41 AM

Hi eikichi91,

Are you going to do regular RT-PCR or qRT-PCR using the primers? That matters because they have different rules.

You can take a look at this old thread for guidance http://www.protocol-...ign-the-primer/. That thread is a bit long, to summarize here, the steps you need are:

1. Find the mRNA sequence for your genes in NCBI nucleoitide database. Since many genes have multiple mRNA sequences deposited in the database, you want to use Refseq for your primer design, because Refseqs represent the most complete and correct sequences for genes. To find the RefSeq for a gene, you can visit the NCBI Gene database to first find the gene.

2. Copy the mRNA sequence and paste it into a primer design program such as Primer3 or NCBI Primer-BLAST to design primer. I recommend Primer-BLAST which uses primer3 at its backend and takes care of everything.

I hope that helps. If you have further question, please let us know.





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