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Primer Blast

primer clustalW BLASt

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6 replies to this topic

#1 Mad researcher

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Posted 12 October 2012 - 07:15 AM

I don't think the title justifies on what am going to ask but i couldn't find anything else:

Assuming that i don't know what size are my primers and on the gel i don't know what size i will have to look for after running the PCR.
I just know that you do it using clustalW by aligning the primer with the genomic DNA. But how do i infer the results?

Can anyone help me please?
Cheers,

Mad Researcher

#2 Trof

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Posted 12 October 2012 - 12:32 PM

You don't know the size of your primers or the size of your product? If you know their sequence you can find the product legth.

The oldest way was probably just blast the primer and find a 100% hit (if you don't even know the exact target sequence) now search for the same accession number on blast results of second primer, there are always numbers of the query sequence, so you take the first and the last and calculate the length.

If you have the exact sequence, you can blast2seq or align with clustal (but will clustal tell you the possition number of align? I'm not sure, bl2seq will. You get the same numbers but you have to substract.

Also finding soem in silico PCR applications can test your primers for specific and other not-so-specific amplicons.

But what I really don't understand is why you call the topic "Primer Blast" and then don't think it's related, when Primer Blast is exactly what will tell you what the product size and it's the easiest way. You wrote an answer to your question to a title.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 Mad researcher

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Posted 12 October 2012 - 01:29 PM

Torf i was bit confused about the topic.

I will try on what you said. You are a life savior Trof :-)
Cheers,

Mad Researcher

#4 Mad researcher

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Posted 12 October 2012 - 01:34 PM

And for those who are viewing this post and would like to know more about in-silico PCR, i found a good reference:


In Silico PCR Analysis

Bing Yu and Changbin Zhang


http://www.springerl...31/fulltext.pdf
Cheers,

Mad Researcher

#5 Mad researcher

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Posted 17 October 2012 - 11:49 PM

http://genome.csdb.cn/cgi-bin/hgPcr

http://insilico.ehu.es/PCR/

I couldn't find rainbow trout genera in these two In silico PCR webpage. How Can i input my own sequence here?
Cheers,

Mad Researcher

#6 Trof

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Posted 18 October 2012 - 02:21 AM

Probably it's not possible, those tools don't use one sequence, but possible whole genomic sequence for comparison.

But PrimerBlast can check your primers against all rainbow trout sequences (not complete, but there are many thousands of them in the GeneBank database), and even agains multiple species.

These in silico PCR tools were fun once, when there was just ordinary Blast, but now PrimerBlast has AFAIK much more capabilities. (It won't tell you how many products you will get in nonspecific temperatures, that's true, but that can be evaluated from the similarity of the sequence and anyway specifity differs even with different buffer conditions and PCR enhancers)

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#7 Mad researcher

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Posted 18 October 2012 - 02:40 AM

Maybe clustalW would help?
Cheers,

Mad Researcher





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