Dear Maitham

on my BLOG: ProteoCool ( https://proteocool.blogspot.com/?m=1) at page 1 you can find:

ProteoCool n°2 (Primer Design: Restiction/Ligation )

ProteoCool n°3 (Primer Design: PIPE Cloning )

i hope that those may be usefull to you.

good luck

ciao

Manuele

Dear colleagues, I prefer Primer3 algorithm (http://bioinfo.ut.ee/primer3-0.4.0/) for gene expression and classic PCR, in the case of allelic discrimination I add IDT oligoanalyzer (https://eu.idtdna.com/pages/tools/oligoanalyzer) to determine the best positions for LNA and for estimation the power of discrimination on the base on dG.

For all cases it is very comfortable to predict reaction effectiveness with "The mfold Web Server" (http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form).

Of course, after you finish design it is crucial to use Blast to prevent at least part unspecific amplification. Unfortunately on real life it is possible to see artefacts that should be fixed by reaction optimization or even by redesign.

Dear, I always use this tool for primer design that would be helpful for you (http://biotools.nubic.northwestern.edu/OligoCalc.html).

Thanks