RobusT™ I RT-PCR Kit

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™ 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
10x 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 30 µg E. coli ribosomal RNA/ml	20 µl	20 µl

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)

Storage and 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 info

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

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