Cloning PCR fragment into the expression vector

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Mar 7 09:40:32 EST 2002


>
>For the PCR product, I have purify through agarose gel to get rid of Taq
>polymerase before doing ligation while the vector,

Products from regular Taq usually are hard to clone, since taq often adds
an extra nucleotide to the end-- usually an A.  This problem can be
overcome (and actually exploited in so called AT vectors) if you just want
to clone the fragment.  However, for an expression construct where frame
might be important, it's just one more headache.  I'm not sure how much of
a problem it is with other thermal stable polymerases, but with plain old
Taq, it's usually been a problem in my experience.  I usually find it
preferable to engineer the desired restriction sites (even if they blunt)
into the primers.


 a double digestion was
>performed with EcoRI and XhoI and Klenow fragment + dNTP was added to fill
>the 5' overhang. Then the 4.9 kb band was purified from agarose gel and
>performed dephosphorylation with Alkaline phosphotase to avoid self
>ligation. The linearised vector was again purified from agarose gel to get
>rid the Alkaline phosphatase (which will inhibit ligation). Ligation process
>was performed  before transformation was done.

This is a lot of manipulation on a gel, and depending on the UV source
you're using, you could be ruining your DNA.  What I would suggest instead
would be to do your digestion.  Then do your alkaline phosphotase treatment
(AP works much better on protruding phosphates than on blunt or recessed
phosphates).  This can usually be done simply by adding the phosphotase to
the restriction digestion.  Phenol extract and precipitate to get rid of
phosphotase, and then do your fill in reaction.  I personally prefer to use
T4 polymerase for fill-ins-- for me it has a better track record than
Klenow.

Hope these suggestions help.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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