RobusT II RT-PCR Kit

RobusT™ II RT-PCR Kit

(M-MuLV RT RNase H^-/DyNAzyme EXT) - A choice for low abundance RNA

Description

RobusT™ II Kit is a high fidelity one-tube system with excellent performance and sensitivity. First-strand synthesis is catalysed by Moloney murine leukemia virus (M-MuLV) reverse transcriptase, RNase H^-. Subsequent second strand PCR is performed by DyNAzyme™ EXT DNA Polymerase, a unique blend of thermostable DNA polymerase and proofreading enzyme.

M-MuLV RT RNase H^- is purified from a recombinant source and due to the absence of RNase H activity it yields much larger quantities of full-length cDNA transcripts than wild type M-MuLV RT, which possesses substantial RNase H activity. RobusT™ II RT-PCR Kit offers an optimal enzyme combination for synthesis of long cDNAs from a variety of RNA sources and target sizes.

Applications

	Detection of gene expression
	Quantification of gene expression
	Generation of cDNA products with high fidelity for cloning and sequencing
	Clinical diagnostics: detection and quantification of infectious micro-organisms, cancer cells and genetic disorders.

Advantages

	High sensitivity of M-MuLV reverse transcriptase, RNase H^-
	Capable of amplifying long targets, up to 6.0 kb
	One-tube system: rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contamination
	Minimal hands-on time required
	Amplifies products from a wide variety of total RNA or mRNA sources
	Saves time by eliminating the need to construct and screen cDNA libraries

Components

The kit contains sufficient materials for 100 (F-590L) or 20 reactions (F-590S) including control reactions.

RobusT™ II RT-PCR Kit
Component	Concentration	F-590S	F-590L

10 x RobusT™ Reaction Buffer		1.5 ml	1.5 ml
MgCl₂ solution	50 mM	1.5 ml	1.5 ml
dNTP mix	10 mM each	20 µl	100 µl
M-MuLV Reverse Transcriptase, RNase H-	5 U/ µl	40 µl	200 µl
DyNAzyme™ EXT DNA Polymerase	1 U/ µl	40 µl	200 µl
Upstream Control primer	0 pmol/µl	20 µl	20 µl
Downstream Control primer	10 pmol/µl	20 µl	20 µl
Control RNA with carrier	10 ng MS2 RNA/µl	20 µl	20 µl
	30 µg E. coli ribosomal RNA/ml

Storage stability

Store all kit components at -20 °C. For long term storage, the control RNA may be stored at -20 °C. The RobusT™ II RT-PCR System is stable for one year from the date of packaging when stored and handled properly.

ORDERING INFORMATION
RobusT™ II RT-PCR Kit	Reactions

F-590S	20 reactions
F-590L	100 reactions

Individual components also available separately
M-MuLV Reverse Transcriptase, RNase H^-	Size	Concentration
F-572S	10,000 U	200 U/µl
F-572L	50,000 U	200 U/µl
DyNAzyme™ EXT DNA Polymerase
with Optimized EXT Buffer and Buffer Pack for Mg²⁺ Optimization	Size	Concentration
F-505S	200 U	1U/µl
F-505L	1,000 U	1U/µl

Figures

Broader working range with RobusT™ II RT-PCR system


M	DNA/Hind III + øX174/Hae III Digest
1	556 bp	human ADP Ribosylation Factor 1 (10 ng human skeletal muscle total RNA)
2	1.0 kb	dystrophin (100 mg human skeletal muscle total RNA)
3	2.2 kb	dystrophin (100 mg human skeletal muscle total RNA)
4	4.1 kb	dystrophin (100 mg human skeletal muscle total RNA)
5	6.0 kb	dystrophin (100 mg human skeletal muscle total RNA)
M		DNA/Hind III + øX174/Hae III Digest

Higher sensitivity for low abundance RNA

		RNA: Human skeletal muscle total RNA
		Target: Human ADP Ribosylation Factor 1
		Marker: DNA/Hind III + øX174/Hae III Digest
		Each successive lane represents decreasing amounts of total RNA from human skeletal muscle ranging from 100 nanograms to 1 picogram.

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