pCDNA3 Cloning Difficulties

[Alexander Kraev]

>I am trying to do basic, straightforward cloning with pCDNA3 and I'm
having a devil of a time getting the thing to ligate (even to itself 
with
only one restriction cut - HindIII).  I've been reasonably careful with
the enzymes (down to 1 U/ug, short digestions, etc.) but I'm still 
getting
no colonies (and yes the transformation is working).  I seem to recall 
an
earlier post where someone else was having similar difficulties.  Anyone
have any suggestions?<

These days there is a very funny cause why ligations produce no
colonies: many use just ONE MICROLITER of ligase and ligate overnight
as "classic" manuals recommend, and...get nothing at all. The likely
reason is that in the first hour of ligation everything was ligated
into a long concatemer which would rarely give a single colony ( and if 
it does, it almost certainly contains a rearranged plasmid). For a 
HindIII ligation one should use 0.1 Weiss unit per 20 ng of a pUC vector 
for an overnight ligation; it is, however, also possible to use 1 unit
and ligate 30 min. In contrast, one rarely comes over smth like
"phosphatase contamination of a restriction enzyme" in 1996.

Happy ligations,

 
 



Alexander Kraev, PhD                     Internet:    
kraev at bc.biol.ethz.ch
> Lab.of Biochemistry III                            Phone:   0041-1-632-31-47
> Swiss Federal Inst. Of Technology           FAX:      0041-1-632-12-13
> Universitaetsstr. 16                    Home Page: http://www.bc.biol.ethz.ch/BiochemistryIII/                                 
> CH-8092 Zurich                                     /Sasha/kraev.html