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C DNASynthesis Kit Protocol

It is a cDNA synthesis protocol

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Maria
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15 views4 pages

C DNASynthesis Kit Protocol

It is a cDNA synthesis protocol

Uploaded by

Maria
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Product Insert

cDNA Synthesis Kit

LOT: See product label EXPIRY DATE: See product label

ORDERING INFORMATION
CAT. NO. SIZE PACKAGE CONTENT

BR0400401 10 rxn of 20 µl 10 µl RevertUP™ II Reverse Transcriptase


100 µl 5× cDNA Synthesis Buffer
20 µl dNTP Mix (10 mM each)
5 µl RNase Inhibitor
10 µl Hexamer Primer
5 µl Oligo (dT) Primer
1.5 ml PCR Grade Water
BR0400402 25 rxn of 20 µl 25 µl RevertUP II Reverse Transcriptase
100 µl 5× cDNA Synthesis Buffer
50 µl dNTP Mix (10 mM each)
12.5 µl RNase Inhibitor
25 µl Hexamer Primer
12.5 µl Oligo (dT) Primer
1.5 ml PCR Grade Water
BR0400403 125 rxn of 20 µl 125 µl RevertUP II Reverse Transcriptase
500 µl 5× cDNA Synthesis Buffer
250 µl dNTP Mix (10 mM each)
62.5 µl RNase Inhibitor
125 µl Hexamer Primer
62.5 µl Oligo (dT) Primer
5 × 1.5 ml PCR Grade Water

COMPONENT COMPOSITION
RevertUP II Reverse RevertUP II Reverse Transcriptase, 200 U/µl in Storage buffer, containing
Transcriptase 50% glycerol
5× cDNA Synthesis Buffer Optimized 5× cDNA synthesis buffer
dNTP Mix (10 mM each) Aqueous solution (pH 7.0) containing 10 mM each: dATP, dCTP, dGTP, dTTP
sodium salts
RNase Inhibitor RNase Inhibitor, 40 U/µl, in Storage buffer, containing 50% glycerol
Hexamer Primer 25 µM Random Hexamer Primer
Oligo (dT) Primer 10 µM Oligo (dT) Primer
PCR Grade Water Ultrapure, sterile filtrated water, DNase-, RNase- and protease-free

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

PIN-04004-002 www.biotechrabbit.com page 1


cDNA Synthesis Kit

FEATURES
· Highly efficient synthesis of long cDNAs (≥ 19 kb)
· Excellent yields at temperatures up to 55°C
· High sensitivity reverse transcription from low abundance template
· Superior performance in demanding applications, including templates with a high degree of secondary
structure

APPLICATIONS
· First-strand cDNA synthesis for RT-PCR and qPCR
· Gene expression profiling
· RNA labeling and primer extension
· cDNA library construction

DESCRIPTION
biotechrabbit™ cDNA Synthesis Kit provides superior components that ensure efficient first-strand cDNA
synthesis from mRNA or total RNA templates. biotechrabbit RevertUP™ II Reverse Transcriptase enables highly
efficient reverse transcription with increased thermostability.
biotechrabbit RNase Inhibitor is a potent non-competitive inhibitor of RNases. The combination of highly efficient
cDNA synthesis, effective RNase inhibition and pure dNTPs allows high yields of cDNAs of more than 19 kb.
For greater application flexibility, hexamer primers, allowing all RNAs in the reaction to be used as templates, and
an oligo (dT) primer, for the synthesis of cDNA from only poly(A) tailed mRNA, are included.

PROTOCOL
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).

page 2 [email protected] PIN-04004-002


cDNA Synthesis Kit
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) 2 µl 1 mM (each dNTP)
RNase Inhibitor, 40 U/µl
0.5 µl 1 U/µl
(optional)
Oligo (dT) 12–18 (10 µM) – or 0.5 µl 0.25 µM
Hexamer Primer (25 µM) – or 1 µl 1.25 µM
Gene Specific Primer (10 µM) 0.5 µl 0.25 µM
5× cDNA Synthesis Buffer 4 µl 1×
0.1–1 µg total RNA or
RNA Template
50–500 ng mRNA (polyA)
RevertUP™ II Reverse
1 µl 10 U/µl
Transcriptase
PCR Grade Water Variable
Total volume 20 µl

· Mix and collect the drops by centrifuging briefly.


· When using
o Hexamer Primer, incubate 10 minutes at 30°C followed by 50–55°C for 20–60 minutes
o Oligo (dT) or gene-specific Primer incubate at 50–55°C for 20–60 minutes.
· Inactivate enzyme 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.

PIN-04004-002 www.biotechrabbit.com page 3


cDNA Synthesis Kit
CERTIFICATE OF ANALYSIS
Quality Control
Functional Assay:
cDNA synthesis with specific primers, followed by quantitative PCR.

Quality confirmed by: Head of Quality Control

SAFETY INSTRUCTIONS
For safety instructions please see Safety Data Sheets (SDS)/Sicherheitshinweise finden Sie in den SDS unter:
http://www.biotechrabbit.com/support/documentation.html.

USEFUL HINTS
· Visit Applications at www.biotechrabbit.com for more products and product selection guides.
· Most biotechrabbit products are available in custom formulations and bulk amounts.

CONTACT BIOTECHRABBIT
biotechrabbit GmbH [email protected]
Volmerstr. 9a [email protected] Phone: +49 30 555 7821-10
12489 Berlin, Germany www.biotechrabbit.com Fax: +49 30 555 7821-99

Legal Disclaimer and Product Use Limitation


Purchase of product does not include a license to perform any patented applications; therefore it is the sole
responsibility of users to determine whether they may be required to engage a license agreement depending upon
the particular application in which the product is used. This product was developed, manufactured, and sold for in
vitro use only. It is not suitable for administration to humans or animals.
Trademarks: biotechrabbit™ (biotechrabbit GmbH).
valid from 24.08.2016

page 4 [email protected] PIN-04004-002

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