You are theoretically right, but you may find that there are many background colonies without inserts since some vector DNA may be cut only once by either of your enzymes. This type of DNA is not separable from double cut molecules and self-relegated with a much higher efficiency than vector/insert ligation. You may find it more efficient to obtain your construct by dephosphorylating your vector and phosphorylating your oligo inserts.
Zhonglin Chai Sent from my iPad On 25/05/2012, at 2:31, "Ed Siefker" <[email protected]> wrote: > I'm trying to clone a short sequence by annealing two complimentary > oligos with incompatible overhangs. Am I correct in understanding > that if I'm doing directional cloning and don't dephosphorylate my > vector, I do not need to phosphorylate the oligos? > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > ______________________________________________________________________ > This email has been scanned by the Symantec Email Security.cloud service. > ______________________________________________________________________ This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please promptly delete this email and notify the system manager. This email has been scanned by the Symantec Email Security.cloud service. For more information please visit http://www.symanteccloud.com _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
