Enzymes for COBRA - (Apr/26/2010 )
This is my first attempt at COBRA. I have been trying to design bisulphite primers for a high density CpG island using methprimer but got no results from the software. So I manually designed them and it seems to work (but gives me a poor yield and is inconsistent, have tried to optimise it but to no avail). The region of interest in the CpG island is based upon BstUI sites since I want to do a COBRA. The software picks up primers downstream of this region (but still in the CpG island). My doubts are:
Are there any other enzymes routinely used in COBRA?
Are primers designed manually besides a software known to work with good efficiency?
What are the factors to keep in mind while designing good bisulphite primers besides those mentioned below?
(Primer length - 20-25bp, similar GC content between primer pair, no primer dimmers or secondary structures, Tm between 55-65, maximum of 1CpG in the primer, Tm difference between primer not to exceed 5C, )
Can I get some suggestions please
forgot to mention that my amplicon size is 330bp
TaqI recognises a methylate bisulfite converted HpaII site.
CCGG > TCGA