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Rt-PCR primer design


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#1 Kiana

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Posted 29 October 2011 - 02:26 AM

Hi,

I have some questions regarding RT-PCR primer design.
This is the way that I think I should use to design primers but I have some difficulties!!

Search the gene of interest in ensemble ---> click on the transcript and select the largest sequence (Is there any other way to get the best sequence ??) --->in supporting evidence click on exons ---> copy all the exons in a word file ---> select a chunk of exonsrandomly and past into Primer3 input ---> get the primers!!

How I can check if the primers are located at the exon-intron boundry?
What is the ref-sequence?
What is the houskeeping gene and why I have to one while performing RT-PCR?
How the get the specific primers, what else should I do?

Thank you very much for your help :)

#2 pcrman

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Posted 29 October 2011 - 04:07 PM

You may want to read this post on RT-PCR primer design http://www.protocol-...ign-the-primer/

It is more appropriate to obtain the RefSeq for the gene from NCBI Gene database and then use the sequence to design primers using a program of your choice such as Primer3. Later, you can impose restrictions on where the primers should be designed for the purpose of including exon-exon junctions.

#3 Curtis

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Posted 29 October 2011 - 07:48 PM

have you tried primer BLAST at NCBI? it's a good tool for beginners. it has options for intron/exon.

#4 pcrman

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Posted 30 October 2011 - 07:50 AM

Yes, Curtis is right. NCBI Primer-BLAST is a very convenient tool which takes care of every thing.

#5 Kiana

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Posted 30 October 2011 - 10:25 AM

Thank you very much for reply :)
I have tried to design my primers from the guidance in this link http://www.protocol-...ign-the-primer/ but still a bit confused about blat and how it works!! I couldnt get the primer at exon-intron boundary, what I have to do to get them right?
about Primer-Blast, can I use to design by itself or i need to do some steps here and then check the primers from other websites?
Is there any primer design program which works just with the gene symble or refseq and design the primers in the exact exon-intron position?

Ill appreciate ur help :)
Many thx :)

#6 pcrman

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Posted 30 October 2011 - 02:05 PM

If you use Primer-blast, you can choose one of the options: span exon junction or intron inclusion, then you don't need to do anything.

#7 Kiana

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Posted 30 October 2011 - 03:50 PM

Thx :)
Should I select primer must span an exon-exon junction in exon junction span or tick the intron inclusion? which one means intron-exon junction?

#8 Kiana

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Posted 31 October 2011 - 10:15 AM

Hi,

I have another question.
My postdoc suggested me to check if the primer is present in all the transcripts. Is there any way to design them considering this as well as intro-exon junction??

Thx




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