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Kits for Molecular Biology

	One-Tube RT-PCR System
	Random Prime Labeling Kit (20 rxns)
	2 x HotStart PCR Master Mix

One-Tube RT-PCR System
Sufficient for 200 Reactions

Description: The one-step RT-PCR kit contains all necessary reagents for high specificity first strand cDNA synthesis and subsequent PCR amplification. TthPlus DNA polymerase is isolated from the Thermus thermophilus strain. TthPlus DNA polymerase is a single 92 kDa polypeptide showing a 5′-3′ exonuclease activity but lacking 3′-5′ exonuclease activity. It catalyzes the polymerization of nucleotides into double-stranded DNA in the presence of MgCl2. Its efficiency has been shown more particularly on large DNA fragments up to 12 kb (using lambda phage DNA as a template). TthPlus DNA polymerase is also capable of catalyzing the polymerization of DNA using a RNA template in the presence of Mn2+. The ability of TthPlus DNA polymerase to reverse transcribe at elevated temperatures (70°C) minimizes the problems encountered with strong secondary structures in RNA since they are unstable at higher reaction temperatures. Higher temperatures also result in increased specificity of primer hybridization and extension. In coupled RT/PCR assays, TthPlus is about 50-100 times more efficient than regular Taq DNA polymerase.

The kit provides a one-tube 10X buffer for both reactions (RT and PCR) using the same enzyme (TthPlus). The 10 x one-tube buffer does not contain Mn(OAc). Mn(OAc) must be added at a final concentration of 3.5 mM if reverse transcription is required.

Application:

	For single tube RT-PCR and Real-Time Quantitative PCR;
	For high temperature reverse transcription that allows highly specific priming of cDNA synthesis and overcomes problems with secondary structure.

Storage: Store at -20°C, in a constant temperature freezer.

Components:

TthPlus DNA polymerase, 500 units

10 x OneTube buffer, 1.5 ml

50 mM Mn(OAc)2, 0.5 ml

Nucleotide Mix (10 mM dATP,

10 mM dCTP, 10 mM dGTP, 20 mM dUTP), 0.25 ml

The optimal experimental conditions depend on the system used and they should be individually determined. The Mn2+ concentrations and the enzyme amount are the limiting factors for an accurate result. Normally 1.5-2.5 units of enzyme and a Mn-acetate concentration of 3.5 mM are used for the reverse transcription in a final 25 µl reaction

Part #	Description	Price ($US)	Quantity
MK-330-0200	200 rxns (25 µl each)	$229.00

- Ordering Instructions -

Random Prime Labeling Kit (20 rxns)
radioactive labeling of DNA

Application: Generation of high specific activity DNA hybridization probes from 10 ng to 3 µg of DNA in a standard reaction. Supercoiled or linearized DNA may be used for labeling DNA of different lengths. Probes can be produced from fragments purified from a variety of sources.

Description: The random prime labeling kit can be used for rapid random-primed labeling of DNA with [³²P], [³²S] or [³H] dCTP to a specific activity. The kit is based on the method described by Feinberg and Vogelstein (1, 2), in which a mixture of random hexamers is used to prime DNA synthesis from any DNA template. In particular this kit may be used to label DNA fragments directly after isolation from agarose gels using the MegaPure GeneClean DNA purification kit.

Components:

20 labeling reactions:

25 µl Klenow fragment (125 units);

100 µl primer mix (100 mM Tris-HCl pH 7.5; 100 mM MgCl₂; 4.5 µM hexamers);

100 µl dNTP mix (5 mM each dATP, dGTP, dTTP, 10 mM DTE).

Storage: Store at -20°C in a constant temperature freezer.

Part #	Description	Price ($US)	Quantity
MK-220-0200	Random Prime Labeling Kit (20 rxns)	$89.99

- Ordering Instructions -

2 x HotStart PCR Master Mix
Hot Start function for all cycles of PCR amplification.

Description: HotStart PCR Ready Mix is a 2 x concentrated reagent mix for Hot Start PCR comprising two cold sensitive mutant Taq polymerases, 2 x PCR-buffer with 5 mM MgCl2, 400µM of each dNTP and stabilizers. Unlike monoclonal antibody based hot start Taq or chemical modified hot start Taq, cold sensitive mutant Taq will retain the hot start ability throughout the whole amplification cycles.

Cold sensitive mutant Taq polymerases are designed for "Hot Start" PCR due to their suppressed activity at low temperature. It offers excellent specificity and two-fold higher fidelity than wild-type Taq. It is designed for PCR with difficult templates such as GC-rich fragments and microsatellites. Cold sensitive mutant Taq polymerases are particularly well suited to primer extension of Single Nucleotide Polymorphism (SNP) markers.

Cold sensitive mutant Taq polymerases maintain excellent specificity and minimal background even in conditions designed for high yield (high Mg²⁺/primer concentrations). In fact, even on genomic templates, the enzyme can be used with MgCl²⁺concentrations as high as 10 mM.

Cold sensitive mutant Taq polymerases are capable of extending through difficult regions, e.g. regions, which include inverted tandem repeats and those with high amounts of secondary structure.

Cold sensitive mutant Taq polymerases work in a totally unique way, involving improved nucleotide selection at the active site, and a much lower rate of mis-match extension, meaning that only perfectly aligned primers will be extended. As a result, the enzyme can give even higher specificity than hot-start (manual or automatic) techniques without the need for inconvenient pre-incubation steps.

Application:

	Hot start PCR amplification of all various templates or tasks ;
	designed for amplification of difficult templates, such as GC-rich fragments and microsatellites;
	primer extension of SNP markers;
	amplification of genomic DNA targets up to 10 kb with high fidelity, specificity, and sensitivity;
	high through-put Hot Start PCR with high specificity, sensitivity, and yield;
	routine diagnostic Hot Start PCR requiring high reproducibility.

	Quality Control: Activity, non-specific endonucleases, nickases, and exonucleases.
	Storage: Recommended to keep at 2-8°C for immediate use (12 month) and -20°C for long term storage.

>> Download PCR Master Mix datasheet

Part #	Description	Price ($US)	Quantity
DP-016-0250	Hot Start PCR Ready Mix (2x), monoclonal antibody based hot start 250 rxns (20 µl each)	$149.00
DP-016-1000	Hot Start PCR Ready Mix (2x), monoclonal antibody based hot start 1000 rxns (20 µl each)	$516.00

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Kits for Molecular Biology

One-Tube RT-PCR System Sufficient for 200 Reactions

Random Prime Labeling Kit (20 rxns) radioactive labeling of DNA

2 x HotStart PCR Master Mix Hot Start function for all cycles of PCR amplification.

One-Tube RT-PCR System
Sufficient for 200 Reactions

Random Prime Labeling Kit (20 rxns)
radioactive labeling of DNA

2 x HotStart PCR Master Mix
Hot Start function for all cycles of PCR amplification.