blunt ligation problems

Steve Lee lees at acpub.duke.edu
Tue Aug 25 17:45:58 EST 1998


In article <199808142224.QAA78238 at nestor.NMSU.Edu>,
Hiranya Roychowdhury <hroychow at NMSU.EDU> wrote:
>
>First modification you should try is to dephosphorylate your vector(s),
>given that your DNA preps are very clean.-

If you used Taq to make the PCR products, they will leave hanging As are
NOT blunt product.. you would have to digest the ends with a nuclease..
or you could use PFU enzyme in your PCR which gives blunt products.  As
said above, dephosphorylating your vector is a must.  Also, you might
try adding some PEG to your ligation mixture as it causes molecular
crowding which is helpful in blunt ligations.

Steve Lee
Loma Linda University
neoender at rocketmail.com




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