Nucleotide Analogs - Jena Bioscience

the polymerase activity as well as RNase H activity,
the 1:1 complex with p51 subunit shows increased
processivity and acivity.
Purity: > 95% by SDS-PAGE.
Store: -20 °C
Selected references:
Pata et al. (2002) Assembly, purifi cation and crystallization of
an active HIV-1 reverse transcriptase initiation complex. Nucl.
Acids Res. 30:4855.
Divita et al. (1995) Dimerization Kinetics of HIV-1 and HIV-2
Reverse Transcriptase – A two step process. J. Mol. Biol. 245:
508.
Mansky et al. (2002) Infl uence of reverse transcriptase variants,
drugs, and Vpr on human immunodefi ciency virus type 1 mutant
frequencies. J. Virol. 77:2071.
Larder et al. (1999) Closing in on HIV drug resistance. Nature
Structural Biology 6:103.
HIV-1 RT, p66 subunit
Recombinant, E. coli
Cat. No. Amount Price (€)
PR-354 10 µg 150,--
Storage buffer: 20 mM KH PO /K HPO (pH 7.1),
2 4 2 4
1 mM DTT, 0.02% Triton X-100, and 50% glycerol.
HIV-1 Reverse Transcriptase is a heterodimer
consisting of phage-encoded p66 and p51 subunit.
The enzyme is used by the Human Immunodefi ciency
Virus-1 to transcribe its RNA genome into DNA. The
RNA-dependent DNA Polymerase I catalyzes the
5’�3‘ synthesis of DNA with RNA template and DNA
primer. The p66 subunit is responsible for polymerase
activity as well as RNase H activity and shows this
activity also in the absence of the p51 subunit. In the
absence of p51, the p66 subunit forms homodimers.
Purity: > 95% by SDS-PAGE.
Standard DNA Polymerase Assay Conditions: The
polymerase activity is assayed in a reaction buffer
containing 50 mM Tris-HCl (pH 8.3), 10 mM MgCl , 2
200 mM KCl, 300 µM dNTPs, and poly(rA)-oligo
(dT) as primer-template. Quantifi cation of RNA-
12-18
DNA duplex is performed using PicoGreen ® .
Absence of contaminants: Tested for the absence of
endo- and exodeoxyribonucleases.
Unit defi nition: One unit is defi ned as the amount of
enzyme that incorporates 1 nmole of dNTPs into acidinsoluble
material in 10 minutes at 37°C.
Store: -20 °C
Selected references:
Tveit H. and Kristensen T. (2001) Fluorescence-based DNA
polymerase assay. Anal. Biochem. 289:96.
Pata et al. (2002) Assembly, purifi cation and crystallization of
an active HIV-1 reverse transcriptase initiation complex. Nucl.
Acids Res. 30:4855.
Divita et al. (1995) Dimerization Kinetics of HIV-1 and HIV-2
Reverse Transcriptase - A two step process. J. Mol. Biol. 245:
508.
Mansky et al. (2002) Infl uence of reverse transcriptase variants,
drugs, and Vpr on human immunodefi ciency virus type 1 mutant
frequencies. J. Virol. 77:2071.
Larder et al. (1999) Closing in on HIV drug resistance. Nature
Structural Biology 6:103.
® Pico Green dsDNA quantitation reagent is a trademark of
Molecular Probes, Inc.
HIV-2 RT
Recombinant, E. coli
Cat. No. Amount Price (€)
PR-352 10 µg 220,--
Liquid. Supplied as 1 mg/ml solution in 20 mM Tris-
HCl, pH 7.8, 1 mM DTT, 1 mM EDTA, 15% glycerol,
and 0.2% BSA.
HIV-2 Reverse Transcriptase (RT) is used by the
Human Immunodefi ciency Virus-1 to transcribe its
RNA genome into single-stranded DNA. HIV-2 RT
can be used for incorporation of nucleotide analogs
into DNA by RT-PCR or as a standard for assaying
HIV-2 RT activity in human serum. In contrast to
HIV-1 RT, HIV-2 RT is not inhibited by non-nucleoside
RT inhibitors (NNRTI´s).
Purity: > 95% by SDS-PAGE.
Activity: 10.000 U/mg (1 unit is defi ned as the amount
of enzyme that incorporates 1 nmol of dTTP into acidinsoluble
products in 10 min at 37 °C)
Store: -80 °C
Selected references:
Pata et al. (2002) Assembly, purifi cation and crystallization of
an active HIV-1 reverse transcriptase initiation complex. Nucl.
Acids Res. 30:4855.
Divita et al. (1995) Dimerization Kinetics of HIV-1 and HIV-2
Reverse Transcriptase – A two step process. J. Mol. Biol. 245:
508.
Mansky et al. (2002) Infl uence of reverse transcriptase variants,
drugs, and Vpr on human immunodefi ciency virus type 1 mutant
frequencies. J. Virol. 77:2071.
Larder et al. (1999) Closing in on HIV drug resistance. Nature
Structural Biology 6:103.
Ren et al. (2002) Structure of HIV-2 reverse transcriptase at
2.35 Å resolution and the mechanism of resistance to nonnucleoside
inhibitors. PNAS 99:14410.
Ligases/Alkaline
Phosphatases/
Nucleotide Kinases
DNA ligases are a large family of evolutionarily
related proteins that play important roles in
a large range of DNA transactions, including
chromosomal DNA replication, DNA repair and
recombination.
Vertebrate cells encode three well-characterized
DNA ligases – DNA ligases I, III and IV –
that appear to be descended from a common
ancestral nucleotidyl transferase enzyme.
Alkaline Phosphatases catalyze the removal
of 5` phosphate groups from DNA, RNA, ribo-
and deoxyribonucleoside triphosphates. They
are a small family of isozymes expressed in
diverse species, including bacteria, mammals,
reptiles, amphibians, nematodes, and insects.
They have been postulated to be involved in
processes such as cell adhesion, vitamin B
transport, metastasis, and cell signaling.
Nucleotide kinases catalyze the transfer of the
gamma-phosphate group from the donor ATP to
the 5’ position of a nucleoside or to the phosphate
moiety of a nucleoside monophosphate
or -diphosphate. Such phosphoryl transfer
reactions are ubiquitous cellular processes for
storage and transfer of chemical energy, and
for controlling the activity of a large variety of
enzymatic systems.
T4 DNA Ligase
Cat. No. Amount Price (€)
EN-149S 15,000 Units 25,--
EN-149L 75,000 Units 100,--
Source: E. coli lambda lysogen NM 989
Description: T4 DNA Ligase catalyzes the formation
of a phosphodiester bond between juxtaposed
5’-phosphate and 3’-hydroxyl termini in duplex DNA
or RNA.
Unit defi nition: One unit is defi ned as the amount
of enzyme required to give 50% ligation of Hind III
fragments of λ DNA (5’ DNA termini concentration of
0.12 µM, 300 µg/ml) in a total reaction volume of 20 µl
Ligases/Alkaline Phosphatases/Nucleotide Kinases
in 30 minutes at 16°C in 1x T4 DNA Ligase reaction
Buffer.
Reaction conditions: 50 mM Tris-HCl (pH 7.8),
10 mM MgCl , 10 mM DTT, 1 mM ATP and DNA (0.1
2
to 1 µM in 5’-termini). Οptimal ligation occurs at 16°C.
Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4),
0.1 mM EDTA, 1 mM DTT, 200 µg/ml BSA and 50%
glycerol.
Store: -20°C.
Heat inactivation: T4 DNA Ligase can be inactivated
by incubation at 65°C for 10 minutes.
Notes:
• One unit is equivalent to 0.015 Weiss units.
• One Weiss unit is defi ned as the amount of
enzyme required to catalyze the exchange of
1 nmol of 32P from pyrophosphate to ATP, into
Norit-adsorbable material in 20 minutes at 37°C.
• T4 DNA Ligase is strongly inhibited by NaCl or
KCl if the concentration exceeds 200 mM.
• Ligation of blunt-ended and single-base pair
overhang fragments requires about 50 times
as much enzyme to achieve the same extent of
ligation as cohesive-end DNA fragments. Bluntend
ligation may be enhanced by addition of PEG
4000 (10% w/v fi nal concentration) or hexamine
chloride, or by reducing the ATP concentration to
50 µM.
To dilute T4 DNA Ligase that will subsequently be
stored at -20°C, 50% glycerol storage buffer should be
used; to dilute for immediate use, 1x T4 DNA Ligase
reaction buffer can be used.
Alkaline Phosphatase,
Calf Intestinal (CIP)
Cat. No. Amount Price (€)
EN-161S 500 Units 40,--
EN-161L 2,500 Units 160,--
Source: Calf intestinal mucosa
Storage buffer: 10 mM Tris-HCl, pH 8.2, 50 mM KCl,
1 mM MgCl , 0.1 mM ZnCl , and 50% glycerol.
2 2
1x Reaction Buffer: 10 mM Tris-HCl (pH 7.9), 50 mM
NaCl, 10 mM MgCl , 1 mM DTT.
2
Alkaline Phosphatase catalyzes the removal of
5` phosphate groups from DNA, RNA, ribo- and
deoxyribonucleoside triphosphates.
It is widely used for the dephosphorylation of
5’-phosphorylated ends of DNA or RNA.
Dephosphorylation also prevents religation of
linearized plasmid DNA in cloning experiments. CIP
can be inactivated by phenol:chloroform extraction, by
gel purifi cation or by spin column.
Purity: > 95% by SDS-PAGE.
Unit Assay Conditions: 1 M diethanolamine-HCl
(pH 9.8), 0.5 mM MgCl , 10 mM p-nitrophenyl-
2
phosphate.
Absence of contaminants: Tested for the absence of
exonuclease, and endonuclease.
Unit defi nition: One unit is defi ned as the amount
of enzyme that hydrolyses 1 µmol of p-nitrophenylphosphate
to p-nitrophenol in 1 minute at 37°C in a
volume of 1 ml.
Specifi c Activity: >2000 U/mg
Store: -20 °C
Selected references:
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, (2nd ed,), 5.72.
Mossner, E., Boll, M. and Pfl eiderer, G. (1980) Hoppe Seylers
Z. Physiol. Chem., 361, 543-549.
Enzymes
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