2 replies to this topic

[catamere]

I want to digest my plasmid with one enzyme and then use an annealed oligo linker to change that enzyme site to another restriction site after the oligo has been ligated into the plasmid.  I know people who routinely use this method, but the oligo design is crucial (and that's where I have the problem).

Will this oligo work to change an PstI site (5' CTGCA>G 3') to a SpeI site (5' A> CTAGT 3')?

Forward:   G ACTAGT
Reverse:      -    TGATCA   ACGT

Thanks for your help!

Edited by catamere, 08 May 2009 - 07:58 AM.

[phage434]

A single 8 base oligo will ligate a SpeI and PstI cut end.   It will destroy both
sites, so that neither enzyme can cut again.  You have to kinase the oligo, or order
it with a 5' phosphate modification.

nnnnnna      ctagtgca        gnnnnnnn
nnnnnntgatc               acgtcnnnnnnn
(SpeI cut)                     (PstI cut)

[catamere]

Thanks for your reply Phage.  That would be fine if I wanted to destroy the site, but I need to CHANGE the site to SpeI from a PstI site.  

What can I do to CHANGE a PstI to a functional SpeI site?  A ds oligo seems to me to be the easier approach.... but a single base off can easily prevent ligation, which is why I need others to review them.

Thanks.