Primer specificity

Cathal Garvey via (by cathalgarvey from
Sat Jul 9 13:10:07 EST 2011

As the primer sequence is all that matters to the PCR reaction, I would use
the primers.

Although I am a stranger to species-specific PCR (and therefore I am not
sure how reliable 18S would be for this task), I imagine you will be trying
to establish which alternative species have the closest match. From that,
calculate a primer annealing temperature below the Tm of the primers but
above the next-best-match. If there are too many alternative matches with
moderate odds of amplifying (ignoring matches that are basically impossible
in your sample), maybe try again with primer design.
On 9 Jul 2011 17:32, "M Aldeeb" <moh_aldeeb from> wrote:
> Dear All,
> I searched for an 18S rRNA gene of an organism on the NCBI website. I
found the sequence and designed primers using the primer tool on the same
website. Now, I want to check the primer specificity (i.e. can I use this
primer as an identification tool to detect a specific species using PCR) and
the question is:
> do I need to BLAST the primer sequence (both forward and reverse) or to
BLAST the sequence of the amplified fragment (PCR product). Many thanks in
> Best regards
> Mohammad
> Email: moh_aldeeb from
> Personal Website:
> --- On Wed, 7/6/11, mnr mnr <mnr475 from> wrote:
>> From: mnr mnr <mnr475 from>
>> Subject: Blunt end cloning
>> To: methods from
>> Date: Wednesday, July 6, 2011, 1:23 PM
>> Hi all.
>> I have tried cloning a potential toxic gene about 1.2 kbp
>> into E.coli with
>> T7 promoter. After two months, I am still not successful. I
>> am now plannig
>> to do cloning into pUC18 vector before subcloning into few
>> other vectors
>> with different promoters. I have limited available
>> restriction sites so that
>> I don't have to redesign PCR primers. Is it feasible to do
>> blunt-end cloning
>> with Phusion polymerase into non-dephosphorylated vectors?
>> Will I only be
>> getting empty vectors after transformations?
>> Thank you.
>> Matt
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>> Methods from
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