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Biotechrabbit

[Biotechrabbit] RNase Inhibitor, 40 U/µl

Cat-No. BR0400901


Biotechrabbit은 바이오의약품 및 생명과학 연구를 위한 고급 실험 재료와 서비스를 제공하는 회사입니다.



제품 설명


RNase Inhibitor, 40 U/µl



제품 번호


BR0400901 - 2500 Units

BR0400902 - 10000 Units


제품 특징


Features

  • Exceptionally pure proprietary Ribonuclease Inhibitor for demanding RNA applications
  • Active under variety of reaction conditions used for work with RNA
  • Prevention of RNA from degradation by a wide range of RNases

Applications

  • In vitro transcription/translation
  • cDNA synthesis
  • RNA purification and storage


Description


biotechrabbit™ RNase Inhibitor is an acidic protein that is a potent inhibitor of a wide spectrum of ribonucleases. The RNase Inhibitor helps to prevent RNA degradation in applications like cDNA synthesis, RT-PCR, in vitro transcription/ translation reactions or RNA purification. The enzyme is purified from a recombinant E. coli strain carrying the RNase Inhibitor gene.


Other Information

Component

Composition

RNase Inhibitor

RNase Inhibitor, 40 U/µl, in storage buffer containing 50% (v/v) glycerol.

 

STORAGE

-20°C (until expiry date – see product label)

 

Unit Definition

One unit is defined as the amount of enzyme required to inhibit 50% of the activity of 5 ng RNase A (hydrolysis of cyclic cytidine-monophosphoric).

Quality Control
Protein Purity

The Protein purity is analyzed by SDS polyacrylamide gel electrophoresis.

RNase Assay

A sample of the enzyme was incubated with a RNA template. RNase activity was not observed after agarose gel electrophoresis.



Protocols

General guidelines
  • RNase Inhibitor can be used in all common reactions performed with RNA, it is active in all common buffers.
  • The recommended final concentration for RNA protection is about 1 -2 units of RNase Inhibitor for every 1 µl of the reaction mixture.
  • Optimal temperature for Ribonuclease Inhibitor activity is 37°C
  • Inactivation conditions: 65°C for 20 minutes
Prevention of cDNA synthesis reaction contamination

RNase contamination is an exceptional concern when working with RNA. RNase A, providing most threat to RNA integrity, is a highly stable contaminant of any laboratory. To prevent RNA from degradation and to minimize possibility of contamination during cDNA synthesis; follow the guidelines below:

  • Use separate clean areas for preparation of the samples and the reaction mixture.
  • DEPC-treat all tubes and pipette tips or use certified nuclease-free labware with aerosol filters.
  • Wear fresh gloves when handling RNA and all reagents.
  • Always assess the integrity of RNA prior to cDNA synthesis in denaturing agarose gel electrophoresis.
  • Use RNase free water and other reagents.
  • To prevent RNA from degradation, add Ribonuclease inhibitor (optional) in to the cDNA synthesis reaction (20 units for 20 µl reaction).
Typical cDNA synthesis reaction set up
  • Thaw on ice and mix very well all reagents.
  • Assemble and keep all reactions on ice.
  • To use time and reagents effectively, always prepare master mix for multiple reactions. For a master mix volume, always calculate the number reactions that you need plus one additional.
  • Combine the following in an RNase-free reaction tube:

Component

Volume

Final concentration

dNTP Mix (10 mM each dNTP)

2 µl

1 mM (each dNTP)

RNase Inhibitor, 40 U/µl

0.5 µl

1 U/µl

Oligo (dT)12–18 (10 µM) – or

Hexamer Primer (25 µM) – or

Gene Specific Primer (10 µM)

0.5 µl

1 µl

0.5 µl

0.25 µM

1.25 µM

0.25 µM

RNA Template

0.1–1 µg total RNA or

50–500 ng mRNA (polyA)

 

Reverse Transcriptase, 200 U/µl
(i.e. BR0400301)

1 µl

10 U/µl

5× RT Buffer

4 µl

1×

RNase-free water
(i.e. BR1900301)

Variable

 

Total volume

20 µl

 

  • Mix and collect the drops by centrifuging briefly.
  • When using
    • Hexamer Primer, incubate 10 minutes at 30°C followed by 50–55°C for 20–60 minutes
    • Oligo (dT) or gene-specific Primer incubate at 50–55°C for 20–60 minutes.
  • Inactivate enzymes at 99°C for 5 minutes.
  • Collect the drops by spinning briefly.
  • Store products at –20°C or proceed to next step, like PCR or qPCR.
  • Use maximum 10 µl of the cDNA synthesis reaction mix for PCR in 50 µl volume.



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