RobusT I RT-PCR Kit

RobusT I RT-PCR Kit

(AMV RT / DyNAzyme EXT) - A choice for problematic RNA

Description

RobusT I Kit is a high fidelity one-tube system. The performance and excellent thermostability of the RobusT I RT-PCR Kit is based on Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT) in the first strand cDNA synthesis and on DyNAzyme^TM EXT DNA Polymerase in the second strand synthesis.

RobusT I RT-PCR Kit offers the best combination of enzymes for amplifying templates with difficult secondary structures: RNA can form various types of secondary structures that are difficult to extend to full-length DNA products. Due to the increased thermostability of AMV RT the cDNA synthesis step can be performed at elevated temperatures up to 70 C, which helps to overcome secondary structure problems. In addition the use of elevated temperatures improves the specificity of reverse transcription by decreasing false priming.

Applications

	Detection of gene expression
	Quantification of gene expression
	Generation of cDNA products with high fidelity for cloning and sequencing

Advantages

	High thermostability of AMV reverse transcriptase
	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
	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-580L) or 20 reactions (F-580S) including control reactions.

RobusT I RT-PCR Kit
Component	Concentration	F-580S	F-580L

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
AMV Reverse Transcriptase	5 U/ µl	20 µl	100 µl
DyNAzyme™ EXT DNA Polymerase	1 U/ µl	40 µl	200 µl
Upstream Control primer	10 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 and AMV Reverse Transcriptase may be stored at -70 °C. The RobusT I RT-PCR System is stable for one year from the date of packaging when stored and handled properly.

ORDERING INFORMATION
RobusT I RT-PCR Kit	Reactions
F-580S	20 reactions
F-580L	100 reactions

Individual components also available separately
AMV Reverse Transcriptase	Size	Concentration
F-570S	500 U	20 U/µl
F-570L	2,500 U	20 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

High thermostability of AMV Reverse Transcriptase

		M
		1-2	60 °C
		3-4	65 °C
		5-6	70 °C

		RNA: Human skeletal muscle total RNA
		Target: Human cytoskeletal gamma actin 625 bp
		Marker: DNA/Hind III + øX174/Hae III Digest
		cDNA synthesis temperature 60°C - 70°C

Broad range of target RNA amplified with RobusT I RT-PCR system


M	*DNA/Hind III + øX174/Hae* III Digest**
1	1.0 kb	MS2 bacteriophage 10 ng RNA
2	1.8 kb	dystrophin (100 ng human skeletal muscle total RNA)
3	3.0 kb	dystrophin (100 ng human skeletal muscle total RNA)
4	5.6 kb	dystrophin (100 ng human skeletal muscle total RNA)
5	6.0 kb	dystrophin (100 ng human skeletal muscle total RNA)

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