Cloning PCR products amplified using Us instead of Ts

Dr Adrian Philbey nswema05 at angis.su.OZ.AU
Fri Jan 19 05:05:50 EST 1996


Us are often used in place of Ts for PCR reactions to allow
Uracil-N-glycocylase to be used for removal of potential PCR
product contaminants. However, PCR products containing Us are
difficult to clone because, upon transformation, bacterial
UNGs destroy the vector plus U-containing insert. The use of
Us instead of Ts for PCR could create problems when a PCR
product turns out to be of interest such that cloning and
sequencing is desirable. In this situation, I have tried to
repeat the PCR using Ts, but on occasion have been thwarted
when I had no more source DNA (from clinical specimens) and
the U-containing PCR product was refractory to being reamplified
using the same PCR primers (or even best-guess nested primers).
To attempt to overcome this problem, I obtained an UNG negative
strain of E coli, CJ236, and transformed competent bacteria with
my U-containing PCR product inserted into a TA vector.
Unfortunately, the plasmids recovered from this strain using
several different methods of plasmid mini-prep appeared to be
degraded.

Does anyone have further suggestions for cloning U-containing
PCR products? It is my opinion that the product information
provided by companies marketing vectors for cloning PCR products
should point out that PCR products containing Us are not
suitable for cloning using UNG positive strains of bacteria.
Do you feel that this potential problem has been adequately
publicised? I can see that considerable time could be wasted
trying to clone PCR products that contain Us using standard
methods.

Adrian W Philbey
Elizabeth Macarthur Agricultural Institute
Camden, New South Wales
Australia

nswema05 at angis.su.oz.au

ps. I repeated this message because I had omitted my address
    from my previous posting and also was not happy with the
    layout. Please accept my apologies.



More information about the Methods mailing list