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Epicentre Forum 6 (1)
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The EZ::TN Insertion Kits can be used for many applications including:
faster sequencing of large DNA molecules than with random subcloning,
primer walking, or generating nested deletions with exonuclease III and
mung bean nuclease; making insertion mutants or gene "knockouts"; and
performing numerous other kinds of experiments using artificial transposons
with different selectable or screenable markers, tags, priming sites,
promoter regions, or a wide variety of structural genes, control elements,
or other sequences.
The EZ::TN™ Insertion System, based on the simple yet elegant
in vitro Tn5 transposition system developed by Goryshin and Reznikoff,1 permits
high efficiency random insertion of an EZ::TN Transposon into any target
DNA molecule in vitro. The EZ::TN System generates insertions more randomly
and at higher efficiencies than other systems. The transposition efficiency
of the EZ::TN System has been increased more than 1,000-fold compared
to wild-type by introducing three different mutations in the transposase
gene and multiple changes in the 19 bp transposase recognition sequence.
The resulting hyperactive transposition system permits one to obtain
transposon insertions even with very large low-copy-number DNA targets
or other DNA targets available in only limited quantities. Target DNA
does not need to be CsCl purified. Recovery of transposition events will
vary depending on factors such as the size of the target DNA and the
transformation efficiency of the competent cells used to recover the
transposon insertion products. As an example, greater than 106 insertions
of the EZ::TN <kan-1> Transposon were obtained per microgram of
a 6 kb plasmid target DNA following transformation of E. coli having
a transformation efficiency of 108 cfu/µg.
EZ::TN™ Insertion Kits
- Goryshin, I. Y. and Reznikoff, W. S. (1998) J. Biol. Chem. 273, 7367.
The MasterAmp™ High Fidelity Long PCR Kit enables consistent amplification
of 40+ kb DNA sequences rapidly and accurately. MasterAmp TAQurate™ Long
DNA Polymerase Mix combines MasterAmp Taq DNA Polymerase with a 3'-->5'
proofreading enzyme to achieve PCR fidelity at least three times better
than Taq DNA polymerase alone. Optimization of difficult, long PCR amplifications
is made easy with the unique PCR optimization premix configuration and
the inclusion of MasterAmp PCR Enhancer (with betaine), which improves
amplification of difficult templates with high GC content or secondary
structure. "Hot start" technologies are not required to obtain PCR amplification
when using this kit in most cases, greatly reducing hands-on manipulation
and cross contamination. The kit includes MasterAmp TAQurate Long DNA
Polymerase Mix and a set of nine pre-optimized 2X Long PCR PreMixes (containing
buffer, Mg2+, dNTPs, and MasterAmp PCR Enhancer) for convenient
and fast PCR set up. A lambda DNA sequence and primers mixture is also
included for control amplification of a 20 kb sequence. Individual MasterAmp™ High
Fidelity Long PCR PreMixes are also available separately. Once the desired
PCR product is obtained from one or more of the nine PCR PreMixes, consistent
amplification may be achieved by purchasing that PCR PreMix individually,
which is sufficient for 200 50-µl PCR reactions.
MasterAmp™ Extra-Long PCR Kit
The MasterPure™ Plant Leaf DNA Purification Kit is designed for
isolating DNA from 35-100 mg of plant leaf tissue and can be scaled up
for larger amounts of tissue. The simple purification procedure, which
can be completed in less than an hour, involves incubating ground plant
leaf tissue in Plant DNA Extraction Solution for 30 minutes at 70°C.
The sample is then chilled and subjected to centrifugation. The DNA is
then precipitated from the supernatant with isopropanol and suspended
in Cleanup Solution. The DNA is then precipitated again with isopropanol.
No enzymatic digestions, CTAB (hexadecyltrimethylammonium bromide), or
organic solvents are used in the MasterPure protocol. The purified DNA
can be used for PCR amplification, restriction digestion, and Southern
blotting.
MasterPure™ Plant Leaf DNA Purification
Kit.
Base Excision Sequence Scanning (BESS) is a powerful method to quickly
and easily obtain T- and/or G-lane sequence data from any PCR product
that is made using labeled primers - without performing dideoxy sequencing
reactions. To do BESS, one simply performs PCR using the BESS dNTP Mix,
treats the PCR product with the BESS reagents, and separates the reaction
products using either manual gels or a capillary- or gel-type automated
sequencer. To perform BESS analysis, it is essential that only one PCR
product is formed from each primer pair for a given template. The BESS
PCR Optimization Kit permits the user to rapidly find PCR conditions
that yield a single PCR product using a set of 12 BESS 2X PCR PreMixes.
Each BESS PCR PreMix contains BESS-optimized dNTPs, buffer, and a range
of magnesium ions and MasterAmp™ PCR Enhancer (with betaine). To
find the optimal BESS PCR conditions, simply perform PCR by adding a
cocktail of your template, primers, and MasterAmp Taq DNA Polymerase
or other Thermus polymerase to each BESS 2X PCR PreMix and cycle. (Proofreading
enzymes like Pfu, Pwo, and Vent DNA Polymerases cannot be used for BESS-T™ analysis.)
Thereafter, always obtain consistent PCR results for BESS analysis by
using the BESS PCR PreMix (available separately) that is optimal for
your primer pair.
The MasterAmp PCR Enhancer* (with betaine) substantially improves
amplification yield and specificity of many target sequences, especially
those containing a high GC content or secondary structure. Betaine lowers
the melting temperature of G+C rich regions to a temperature more
similar to A+T regions. This results in destabilization of double-stranded
regions, which limits polymerase pausing, thereby increasing amplification
yields. The MasterAmp PCR Enhancer (with betaine) can be used with a
wide variety of DNA polymerases.
*Covered by German Patent No. DE4411588C1 and other patents pending in the
U.S. and other countries.
MasterAmp™ PCR Enhancer
The Dark Reader™ Transilluminator is a visible light transilluminator
for the detection of fluorescently stained nucleic acids in gels. Because
the Dark Reader Transilluminator uses visible light, researchers no longer
need to use hazardous ethidium bromide and UV light. The Dark Reader
Transilluminator also eliminates UV damage to DNA, so DNA fragments isolated
from gels maintain integrity for use in downstream applications such
as cloning and sequencing (Table 1). The Dark Reader Transilluminator
allows the direct detection of as little as 100 pg of SYBR® Gold-stained
DNA per band. When used with SYBR Green, the Dark Reader will directly
detect approximately 0.5-1 ng of DNA per band. By using longer camera
exposures, even lower amounts may be detected on film. The gel looks
exactly like an ethidium bromide-stained gel when viewed with the filters
supplied. Photos may be taken using Polaroid film or digital imaging
systems in the same manner as for ethidium bromide-stained gels. The
Dark Reader Transilluminator includes both blue and orange filters necessary
for visualizing stained gels. Two orange filters are supplied: a flat
sheet for placing over the gel for photography and the other as a pair
of glasses to use for hands-free isolation of nucleic acid bands from
gels. Additional Dark Reader Glasses are available separately.
| T7 phage DNA |
UV (360nm), 30 sec. |
1.0 X 104 pfu/ug insert |
| T7 phage DNA |
Dark Reader, 30 sec. |
2.2 X 106 pfu/ug insert |
| Wheat germ DNA |
UV (360nm), 30 sec. |
2.0 X 104 cfu/ug insert |
| Wheat germ DNA |
Dark Reader, 30 sec. |
1.7 X 106 cfu/ug insert |
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