Direct Cloning of Blunt-end PCR Fragments. HELP !

Frosty Loechel pulm012 at uabdpo.dpo.uab.edu
Sat Jul 2 07:19:27 EST 1994


In Article <2v1026$s69 at mserv1.dl.ac.uk>, mjones at crc.ac.uk (Dr. M.D. Jones)
wrote:

>
>I am trying to clone blunt-ended PCR fragments into SmaI cut M13 vectors.
>So far no luck.  Using the same batch of vector, T4DNA ligase and competent
>cells, I can successfully clune AluI digested lambda DNA fragments.
>
>The PCR fragments are 300 - 2000 bp in size.  I clean them up by PEG pption.
>This is a great method and we use it all the time to clean-up PCR fragments
>for ABI DYE-terminator cycle sequencing.  The original reference to this 
>method is Rosenthal et al. (1993) Nucleic Acids Res. vol. 21. 173-174.
>
>The PCR fragments can be digested with restriction enzymes.  I blunt-end the
>fragments with T4DNA polymerase (+/- Klenow).
>

Are you trying to clone your PCR fragments into a phosphatased vector?  If
so, you must put 5' phosphates on your PCR fragments, either by using
phosphorylated oligos to do PCR, or by kinasing your fragments after PCR.
(Or by cutting the ends of the PCR fragment with restriction enzymes.)


Frosty (who admits to having made this mistake himself)



More information about the Methods mailing list