digesting out restriction site - (Aug/17/2006 )

Hi all,
I would like to get rid of BamHI site from my plasmid for that I am going to do double digestion with BamHI and Eco RI, gel purify and religate the plasmid. Hope I will get few colonis and without BamHI site. Please comment to this idea.
Thanks

-sse-

QUOTE (sse @ Aug 17 2006, 02:18 PM)

You can block it probably by methylation (have no experience so I don't know if it works for this enzyme).

-hobglobin-

or could anyone please suggest a plasmid without BamHI and NcoI site?

-sse-

if the two enzymes sticky ends are compatible you should be able to religate and destroy both original sites. In this case you can't because bam and eco have differing overhangs.

Try to find another site that is compatible with bam or eco. Or if you can only use bam and/or eco, chew back (or fill in) the overhang(s) and blunt end religate.

-vasussci-

u have to blunt end the plasmid with klenow, after digestion with both the enzymes or else u would have incomaptible ends and the plasmid will not religate.

-scolix-

QUOTE (scolix @ Aug 17 2006, 02:38 PM)

u have to blunt end the plasmid with klenow, after digestion with both the enzymes or else u would have incomaptible ends and the plasmid will not religate.

Double digest, Mung Bean treatment, and blunt end ligation.

-Matt

-MisticMatt-

definitely go for a blunt ending...
of course, dephosphorylation is not recommended

-fred_33-