Help!!! liagtion problem

Tim Barnett t.barnett at path.utas.edu.au
Tue Jun 11 23:00:14 EST 1996


In article <31BC6A5C.262C at cygnus.tamu.edu>, pettway at cygnus.tamu.edu wrote:

I would try digesting your mtDNA and vector for only an hour.  O/N
digestion would allow any exonuclease contaminants to damage your sticky
Pst I ends.  This would not only result in reduced ligation efficiency but
may also cause any self-religated vector to appear as white colonies.  If
this is the case then your religated vector control will also appear
white, assuming they were plated on X-gal/IPTG plates.  You don't really
need to treat your insert DNA with RNase since RNA won't clone anyway. 
You are also using way too much ligase.  I use 1.5U in each ligation with
500ng of vector.  Do you phosphatase your vector?  If so you may be using
too much which also has exonuclease activity.  You only need 0.1U/pmol of
5'P overhangs.  One final note.  You say the past 8 times you have
attempted this you get 100s of white colonies but none of these contain
mtDNA inserts.  How do you know this?  Have you isolated any of the
plasmid DNA from these colonies and checked Pst I digests on gels?  If so,
where are your inserts coming from if you started with mtDNA in the first
place?  You may have a contamination problem.

Hope this helps,

Tim

-- 
Anthony Chan

e-mail address  af_chan at postoffice.utas.edu.au



More information about the Methods mailing list