PCR product cloning

Matthew L. Brown, Ph.D gemini3 at mindspring.com
Wed Apr 23 16:27:41 EST 1997


Connie,
	Here's another vote for the TA cloning kit from Invitrogen. I use it
routinely and have good results for most experiments. When it hasn't
worked I've attributed it to two possibilities. First I usually use a
proofreading blend of enzymes for PCR like Advantage Klentaq from
Clontech (I do mostly long distance PCR). If I don't remove the
reactions from the thermocycler immediately following the last extension
I have the cycler go to 4 C and hold the samples. Sometimes when I try
to set up the ligation with such a held mixture it either fails or is
poorly efficient. I think the klenow fragment must proofread some (i.e.
remove A overhangs even at 4 C. Just a theory.) Moral: Especially when
using a proofreading blend, use fresh products shortly after extension
step and gel analysis. This ensures the maximum number of molecules
containing A overhangs. Second, If there is even a faint nonspecific
product present in the reaction on a gel this product will usually be
the most efficiently ligated and therefore the most efficiently
transformed. If the product is particularly small the resulting plasmids
with small inserts will lead to a blue or light blue colony when
screened. Moral: Use the most stringent PCR conditions possible to
produce cDNA's. If faint bands are still present purify the product
using extreme care (see my post Rafael, Klaus, and Wolfgang.) It may be
necessary to perform an A-overhang reaction (see Invitrogen manual or
e-mail for details) on the purified product as I expect the A overhangs
are somewhat unstable and this has led to some failed ligations
following purification in my experience. Anyway, best of luck.

Matt Brown, Ph.D.
Research Fellow
Dept. of Medicine
Univ. of Alabama at Birmingham



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