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EPICENTRE Forum 2 (1)
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Lambda in vitro packaging extracts are used for
constructing cDNA libraries, for cloning genomic DNA into lambda and
cosmid vectors, and for rescuing lambda shuttle vectors from transgenic
animal systems. The success or failure of all of these experiments depends
on the efficiency of the extract in packaging cos-site-containing
DNA.
Figure 1 presents the side-by-side packaging efficiency
results obtained using Epicentre's new MaxPlax Packaging Extract with
bacteriophage lambda control DNA compared to results obtained with the
same DNA using a leading, traditional packaging extract from another
supplier (Stratagene). As seen, packaging efficiencies routinely obtained
using the MaxPlax Packaging Extract were consistently higher than the
efficiencies obtained using the traditional extract.
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Figure 1. Representative DNA packaging efficiencies with MaxPlax
Extract or Gigapack III Gold Extract (Stratagene, La Jolla, CA). For
an accurate comparison, two different [lambda]-DNA samples were
used: (A) The control DNA supplied with the Gigapack Gold product,
or (B) the control DNA supplied with the MaxPlax kit. Packaging
efficiency was higher for each DNA using MaxPlax Extract. |
Traditional lambda packaging extracts are derived from
two complementary lysogenic E. coli strains, BHB2690 and BHB2688,
as described by Hohn.1 The MaxPlax
Packaging Extract is a single-tube, ultra high efficiency system that
utilizes a new packaging strain, NM759, recently reported by Gunther,
Murray and Glazer.2 This strain,
which replaces strain BHB2690 in the preparation of the sonication extract,
is a restriction-free, K12-derived strain deficient in the production
of [lambda]-phage capsid protein D. When combined with the complementary
freeze-thaw extract from strain BHB2688,1 which
is deficient in the production of [lambda]-phage capsid protein E, an
extremely high efficiency lambda packaging extract is obtained.
Similar to what was described by Gunther et al.,2 MaxPlax
Packaging Extracts can be used to package methylated genomic DNA. The
ability to package [lambda]-DNA bearing the mammalian methylation pattern
is greatly enhanced in extracts made using the MaxPlax strains, as shown
by the fact that the efficiency of [lambda]-vector rescue from transgenic
mouse DNA was 10- to 20-fold greater than that obtained using extracts
from the BHB2690/ BHB2688 strains and 600-fold greater than obtained
using extracts from E. coli C strains.2 The
lack of restriction activity was crucial for the high efficiency rescue
of lambda shuttle vectors from transgenic mouse DNA.2,3
Every lot of MaxPlax Packaging Extract is optimized for
high efficiency packaging of both methylated genomic DNA and unmethylated
DNA. The economical MaxPlax Extract is available as a convenient, easy-to-use
1-tube reagent, so there is no need to mix different packaging components
together before use. Simply combine the DNA with the extract and plate
the recombinant phage. MaxPlax Kits containing either 5 or 10 1-tube
extracts are available. Each MaxPlax Kit also contains control ligated
[lambda]-DNA, control host bacteria, a complete protocol and a Certificate
of Analysis showing actual packaging efficiency (pfu/µg DNA) obtained
for that lot of MaxPlax Packaging Extract.
- Hohn (1979) Methods Enzymol. 68: 299.
- Gunther et al. (1993) Nuc. Acids Res. 21: 3903.
- Kohler et al. (1990) Nuc. Acids Res. 18: 3007.
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