(by moh_aldeeb from yahoo.com)
Fri Jul 8 23:16:03 EST 2011
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 advance.
Email: moh_aldeeb from yahoo.com
Personal Website: http://sites.google.com/site/draldeebsite/
--- On Wed, 7/6/11, mnr mnr <mnr475 from gmail.com> wrote:
> From: mnr mnr <mnr475 from gmail.com>
> Subject: Blunt end cloning
> To: methods from magpie.bio.indiana.edu
> 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.
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