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Finding upstream sequence and e-PCR


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#1 422C2

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Posted 18 July 2014 - 09:13 AM

Hello experts,

 

I am doing a small summer project in C. elegans lab and in the first step, I am supposed to find out upstream sequence of a few genes and design primers to amplify that region. Once the primers are designed, I am asked to check if those primers bind to any other region of the genome.

 

Can anybody direct me on how to find the upstream region of the gene on Wormbase or on any other database. From what I noticed, Wormbase only gives the sequence of the gene and not the upstream region. Please correct me if I am wrong.

And how do I find out that my primer pair is not going to produce any other product BEFORE running a PCR?

 

Thank you very much.



#2 pito

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Posted 19 July 2014 - 11:19 AM

why not just a primer that binds the start of the gene?

 

Anyway; you can use a website like NCBI (blast) to find genes with the introns or upstream parts of the genes present....

 

Wormbase itself: I do not know this.

 

And whether your primers will give another product depends on the fact they would bind on other genes too.. But to answer this we need to know exactely what you are doing prior to the PCR. Is it genomic DNA? or?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 pcrman

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Posted 20 July 2014 - 11:18 PM

In this post, I provided steps on how to find upstream (promoter) sequences. The instructions apply to worm upstream sequences too.

 

Hope that helps.



#4 422C2

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Posted 21 July 2014 - 01:30 AM

In this post, I provided steps on how to find upstream (promoter) sequences. The instructions apply to worm upstream sequences too.

 

Hope that helps.

I tried to go like that but I did not find an option to find upstream region and where I thought it will give me upstream region, it suggested me to enter flanking region (which I am not aware of). It will be great if you could check the steps with the current release and make sure that the options are still there.

Ensembl is overwhelming

 

@pito,

Yes, I am using genomic DNA and as I have been told that there are some programs that run PCR in-silico on whole genomic region and tells you where it 'may' bind. Not too sure how BLAST will give me that as one primer may bind it but other won't and it is quite a lot of work to check that the both primers will bind in the nearest region to allow PCR product to form.






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