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PCR, qPCR, RT-PCR & dNTPs

RevertAid™ First Strand cDNA Synthesis Kit

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Catalog#	Size, concentration	Certificate of Analysis	MSDS

K1621	for 20 react.	K1621
K1622	for 100 react.	K1622

Product information

Features

Full-length first strand cDNA up to 13 kb.
Increased reaction temperatures in the range of 42-50°C.
Supplied with the recombinant RiboLock™ RNase Inhibitor.
Complete – oligo(dT)₁₈ and random hexamer primers included with the kit.

Applications

First strand cDNA synthesis for RT-PCR and real-time RT-PCR (1, 2).
Construction of full length cDNA libraries.
Generation of probes for hybridization.
aRNA synthesis.

Description

The RevertAid™ First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit is suitable for synthesis of cDNA up to 13 kb.

The kit uses RevertAid™ Reverse Transcriptase which has lower RNase H activity, compared to AMV reverse transcriptase.

The recombinant RiboLock™ RNase Inhibitor, supplied with the kit, effectively protects RNA template from degradation. It is fully compatible with reverse transcription reaction, as it maintains activity at temperatures up to 55°C.

The kit is supplied with both oligo(dT)₁₈ and random hexamer primers. The oligo(dT)₁₈ anneals selectively on the poly(A) tail of mRNA. Random hexamer primers do not require the presence of poly(A). Therefore, they can be used for transcription of the 5'-end regions of mRNA or cDNA synthesis using RNA without poly(A) tail e.g. micro RNAs. Gene-specific primers may also be used with the kits.

The first strand of cDNA can be directly used as a template in PCR (see RT-PCR protocol), real-time PCR or in second strand cDNA synthesis (see protocol).

Quality Control

The kit is functionally tested in RT-PCR using 100 fg control GAPDH RNA and GAPDH control primers generated a 496 bp product visible on agarose gel after ethidium bromide staining.

Components

RevertAid™ Reverse Transcriptase
RiboLock™ RNase Inhibitor
5X Reaction Buffer
dNTP Mix,10 mM each
Oligo(dT)₁₈ Primer
Random Hexamer Primer
Control GAPDH RNA
Forward GAPDH Primer, 10 µM (5'-CAAGGTCATCCATGACAACTTTG-3')
Reverse GAPDH Primer, 10 µM (5'-GTCCACCACCCTGTTGCTGTAG-3')
Water, nuclease free
Detailed Protocol

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Guidelines to Avoid RNase Contamination

RNA purity and integrity is essential for synthesis of full-length cDNA, which results in high quality RT-PCR products. Therefore, RNase contamination is always a concern when working with RNA. The RNA quality can be affected by RNase A, which is a highly stable contaminant of any laboratory environment. All components of the kit have been rigorously tested to ensure that they are RNase free. To prevent contamination both the laboratory environment and all prepared solutions must be free of RNases.

DEPC-treat all tubes and pipette tips to be used in the cDNA synthesis or use certified nuclease-free labware.
Use pipettes dedicated for RNA work.
Wear gloves when handling RNA and all reagents, as skin is an common source of RNases. Change gloves frequently.
Use certified reagents, including high quality water (e.g., nuclease-free or DEPC-treated Water).
Use an RNase inhibitor, such as RiboLock™ RNase Inhibitor, to protect template RNA.
Always assess the integrity of RNA prior to cDNA synthesis. For example, if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total RNA, the mRNA in the sample is considered to be intact.

References

Schmidt, A., Su, Y.H., et al., UPS1 and UPS2 from Arabidopsis Mediate High Affinity Transport of Uracil and 5-Fluorouracil, The Journal of Biological Chemistry, vol. 279, 43, 44817-44824, 2004.
Papavinasasundaram, K.G., et al., Deletion of the Mycobacterium tuberculosis pknH Gene Confers a Higher Bacillary Load during the Chronic Phase of Infection in BALB/c Mice, Journal of Bacteriology, Aug., 5751-5760, 2005.

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