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RevertAid^™ First Strand cDNA Synthesis Kits* 
* Not available in the USA

RevertAid™ H Minus First Strand cDNA Synthesis Kit
#K1631	for 20 reactions
#K1632	for 100 reactions
Related Documents (in pdf, ~50-60 KB): Detailed Protocol: #K1631, #K1632 MSDS (English) MSDS (English-USA) MSDS (German) Flyer QuickProtocol™
RevertAid™ First Strand cDNA Synthesis Kit
#K1621	for 20 reactions
#K1622	for 100 reactions
Related Documents (in pdf, ~50-130 KB): Detailed Protocol: #K1621, #K1622 MSDS (English) MSDS (English-USA) MSDS (German) Flyer

 

Features

• Higher yields of the first strand of full-length cDNA with RNA templates up to 13 kb.
• Wide selection of primers: oligo(dT)₁₈, random hexamer and gene-specific.
• Increased reaction temperatures - the first strand of cDNA can be synthesized within the 42-55°C temperature range.
• Supplied with the recombinant RiboLock™ RNase Inhibitor.

RevertAid™ H Minus First Strand cDNA Synthesis Kit, flyer in pdf, 130 KB

Description
The kits are complete systems for efficient synthesis of first strand cDNA from RNA templates. Each kit uses a different genetically engineered version of the Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase.
The RevertAid™ M-MuLV Reverse Transcriptase has low RNase H activity, compared to AMV reverse transcriptase.
The RevertAid™ H Minus M-MuLV Reverse Transcriptase has a point mutation that completely eliminates RNase H activity.
Both kits are suitable for synthesis of cDNA as long as 13 kb. Greater yields of full-length cDNA from long templates are obtained with the RevertAid™ H Minus M-MuLV Reverse Transcriptase because this enzyme does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cDNA, and it maintains activity over a wider temperature range (42-55°C).
The recombinant RiboLock™ RNase Inhibitor, supplied with the kits, is fully compatible with reverse transcription reaction, as it maintains activity at higher temperatures (up to 55°C) within the pH 5-9 range.
The kits are 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. 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 Protocol for PCR with Taq DNA Polymerase) or in second strand cDNA synthesis (see Protocol for Second Strand cDNA Synthesis).

Applications

• First strand cDNA synthesis for RT-PCR (1, 2).
• Construction of cDNA libraries.
• Generation of probes for hybridization.
• aRNA synthesis.

Quality Control
The kits are functionally tested in synthesis of first strand cDNA using a polyadenylated RNA transcript as a control template with the following set of primers: a specific primer, an oligo(dT)₁₈ and a random hexamer.

Components of the Kit (#K1631, #K1632)

• RevertAid™ H Minus M-MuLV Reverse Transcriptase
• RiboLock™ RNase Inhibitor
• 5X Reaction Buffer
• dNTP Mix, 10 mM each
• Oligo(dT)₁₈ Primer
• Random Hexamer Primer
• Control RNA
• Control Primer
• DEPC-treated Water
• Detailed Protocol (in pdf, ~60 KB):
  • #K1631
  • #K1632

Components of the Kit (#K1621, #K1622)

• RevertAid™ M-MuLV Reverse Transcriptase
• RiboLock™ RNase Inhibitor
• 5X Reaction Buffer
• dNTP Mix,10 mM each
• Oligo(dT)₁₈ Primer
• Random Hexamer Primer
• Control RNA
• Control Primer
• DEPC-treated Water
• Detailed Protocol (in pdf, ~58 KB):
  • #K1621
  • #K1622

Two-step RT-PCR using the RevertAid™ H Minus First Strand cDNA Synthesis Kit and the Long PCR Enzyme Mix.
1 µg of total mouse heart RNA and oligo(dT)₁₈ primer were used in the reverse transcription reaction with RevertAid™ H Minus First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template in subsequent PCR with Long PCR Enzyme Mix. A PCR primer specific to the 5'-end of mouse dystrophin gene mRNA and a series of flanking primers were used to amplify different regions of the gene.
M1 - GeneRuler™ DNA Ladder Mix
1-6 - RT-PCR products
M2 - Lambda – pUC Mix Marker

Related Products

Taq DNA Polymerase (recombinant) Taq DNA Polymerase (native) PCR Master Mix (2X) Long PCR Enzyme Mix High Fidelity PCR Enzyme Mix dNTP Mixes dNTP Set RNase H, E.coli DNA Polymerase I, E.coli T4 DNA Polymerase T4 DNA Ligase Rapid DNA Ligation Kit RiboLock™ RNase Inhibitor DNase I, RNase-free Oligo(dT)18 Primer Random Hexamer Primer Aminoallyl-dUTP DEPC-treated Water

References

1. 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.
2. 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.

Updated balandžio 24, 2008 18:56