| Easy Cloning Simplifies Library Construction
We commonly use error-prone PCR to create
random mutant libraries, which you can subsequently
screen to identify mutants with altered
or improved properties. Amplicons generated
by error-prone PCR can prove more difficult to
clone due to low product yields, mutations at
the ends that interfere with restriction-based
cloning, and/or inefficient synthesis of 3' dA
overhangs or blunt ends, which reduces the
efficiency of TA- or blunt-end cloning strategies.
With current methods, randomizing and
sub-cloning a portion of a gene that encodes a
functional domain can be tedious and time
consuming as there are rarely convenient
restriction sites available. To address this
need, we developed the GeneMorph® II
|
EZClone domain mutagenesis
kit*, which features
an efficient, flexible three-step cloning
method that doesn't require sub-cloning or
specific restriction enzymes.
As illustrated in Figure 1, we first amplified
the domains to introduce random mutations
using error-prone PCR with Mutazyme® II DNA
polymerase. Purified PCR products serve as
mega primers for the EZClone reaction. They
are denatured and annealed to the original
donor plasmid and then extended with a specialized
high-fidelity DNA polymerase to minimize
unwanted errors during the cloning
process. You cycle the reaction several times
before treating it with Dpn I enzyme to remove
background DNA prior to transformation into
competent E. coli. After transformation, these
colonies are ready-to-go for further analysis in
your functional assay. With the entire process
completed in a day plus an overnight transformation,
you save valuable research time that
would have been wasted on trying to find,
introduce and verify unique restriction sites to
excise these domains.
|
A More Uniform Spectrum
Like the GeneMorph® II random mutagenesis
kit, our new GeneMorph® II EZClone kit also
features Mutazyme® II DNA polymerase, which
produces mutation rates from 1 to 16 mutations/
kb, introduces all possible mutation types,
and provides equivalent mutation rates at As
and Ts compared to Gs and Cs (Figure 2). As a
result of reduced mutational bias, libraries created
with Mutazyme II DNA polymerase should
exhibit greater representation compared to
libraries generated with other error-prone PCR
enzymes.
Discover More, Faster
With reduced bias compared to error-prone
PCR methods employing Taq DNA polymerase,
the GeneMorph II EZClone kit offers a fast and
easy method for cloning. It is the ideal choice
for creating large, diverse mutant collections
(Table 1). Screening more representative
libraries allows you to rapidly uncover key sites
responsible for structure-function relationships
and to accomplish your research goals faster
than ever before.
REFERENCE
* See license reference 3
|
