PCR aim band size

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jasmina
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PCR aim band size

Post by jasmina » Tue Apr 06, 2010 8:27 am

hi there!
I want to know how to get a aim band size using BLAST, when 2 primer bands and aim gene name are given.
is there anyone who can help me? thank you O(∩_∩)O~

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JackBean
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Post by JackBean » Tue Apr 06, 2010 8:40 am

just download the sequence of your gene and look, where the primers anneal and from that calculate the size of your amplicon.
E.g. if your fw primer anneals to +25 nt and rev primer anneal to +450, than your amplicon will have 450-25 = ~425 nt
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Post by jasmina » Tue Apr 06, 2010 8:58 am

JackBean wrote:just download the sequence of your gene and look, where the primers anneal and from that calculate the size of your amplicon.
E.g. if your fw primer anneals to +25 nt and rev primer anneal to +450, than your amplicon will have 450-25 = ~425 nt



but in most cases, primers are small, sometimes they can anneals to several site, what should i do in this case? :?

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JackBean
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Post by JackBean » Tue Apr 06, 2010 9:39 am

the primers should be long enough to be quite unique at least for your species.
At PubMed's BLAST is some tool for designing primers
http://www.ncbi.nlm.nih.gov/tools/prime ... =BlastHome
but I have never used it. Anyway, if you already have your primers, just use BLAST to see, where can it anneal.
Also, as you use two primers, you highly decrease the probability of inspecific amplification
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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MrMistery
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Post by MrMistery » Tue Apr 06, 2010 8:18 pm

if your primers anneal to multiple places, then that's a problem in itself.
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

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Post by jasmina » Wed Apr 07, 2010 1:13 am

Thank you ! O(∩_∩)O~ i get it. first download aim gene mRNA sequence ( select genus) ,then blast 2 primers and mRNA sequence with blastn-suite respectively, after get annealing site, calculate band size, right?

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