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1-Step RT-PCR Kit

1-Step RT-PCR Kit

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Published by Syed Mazhar Ali

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Published by: Syed Mazhar Ali on Jun 18, 2012
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06/18/2012

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G-Biosciences, St Louis, MO. USA
 
1-800-628-7730 
1-314-991-6034 
 technical@GBiosciences.com  
 A Geno Technology, Inc.
(USA) brand name
 
think proteins! think G-Biosciences! www.GBiosciences.com
382PR
01
1-Step RT-PCR Kit
INTRODUCTION
The 1-Step RT-PCR Kit is designed for optimal convenience in carrying out highly sensitive and specific RT-PCR in a singletube. 1-Step RT-PCR is a variation on standard (two-step) RT-PCR in which all components of the RT and PCR are mixed inone tube prior to starting the reactions and in which RT and PCR are thus carried out sequentially in one tube. This approachoffers tremendous convenience when applied to analysis of single targets from multiple samples of RNA. This approach alsominimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps, since the RT andPCR reactions are carried out sequentially, without opening of reaction tubes between steps.The kit is composed of high quality reagents. The AMV reverse transcriptase, from Avian Myeloblastosis Virus, is used forRT, and the optimized Taq DNA polymerase is used for PCR. A unique buffer, 10×RT-PCR buffer suitable for both the RTand PCR steps and crucial for successful 1-Step RT-PCR, is included, along with dNTPs mixture and Ribonuclease Inhibitor.DEPC-treated water is also provided. Use of this kit and the accompanying protocol will ensure excellent results.
ITEMS SUPPLIED Cat. # 786-813Part. # Description Size
 
219A-B AMV Reverse Transcriptase [25U/µl] 125U003T-I Taq DNA Polymerase [5U/µl] 125U068R-A RT-PCR Buffer [10X] 50µl210D-A dNTPs [10mM each] 25µl079R-B Ribonuclease Inhibitor [40U/µl] 300U025D-F DEPC-treated water 0.5ml
STORAGE CONDITIONS
The kit is shipped on blue ice. Upon arrival, store
 
at -20°C. This product is stable for 1 year at -20
°
C.The AMV Reverse Transcriptase is supplied in 200mM potassium phosphate (pH7.2), 2mM DTT, 0.2% Triton X-100, 50%Glycerol.
SOURCE
 
AMV Reverse Transcriptase: Avian Myeloblastosis Virus
 
Taq DNA Polymerase: The polymerase gene from Thermus aquaticus (strain YT1) is cloned and expressed in
 E. coli
,then highly purified to produce Taq DNA polymerase.
UNIT DEFINITION
AMV Reverse Transcriptase: One unit is defined as the amount of enzymes that incorporate 1nmol of dTTP into acid-insoluble precipitate in10 min using poly(A)
+
RNA and oligo(dT) as the template/primer at 37°C.Taq DNA Polymerase: One unit (U) of Taq polymerase is defined as the amount of enzyme needed to catalyze theincorporation of 10 nanomoles of deoxyribonucleotides into acid-insoluble material in 30 minutes at 70°C using herringsperm DNA as a substrate.
 
Page
2
of 
3
 
IMPORTANT INFORMATION
 
Ensure the integrity and purity of your RNA. The quality of RNA is the key for first-strand cDNA synthesis. Theintegrity and purity of RNA can be inspected by the ratio of OD
260
 /OD
280
and agarose gel analysis. The common ratio of purified RNA is 1.8~2.0, if not, the RNA should be purified further. The ratio of eukaryotic RNA 28S/18S is about 2:1,if not, the RNA has been degraded.
 
Avoid RNase contamination. All vessels, reagents and solutions must be sterile, and all procedures must be performedwith gloves.
PROTOCOL
1.
 
Add the following components into 0.2ml or 0.5ml thin-wall PCR tube:
Reagent Volume
Total RNA 0.5~1µgForward primer 10~30pmolReverse primer 10~30pmolRT-PCR Buffer [10X] 2µldNTPs [10mM each] 1µlAMV Reverse Transcriptase [25U/µl] 0.2µlTaq DNA Polymerase [5U/µl] lRibonuclease Inhibitor [40U/µl] 0.25µlDEPC- treated water up to 20 µl2.
 
Mix the solution and centrifuge briefly, then incubate for 30 min at 45°C.3.
 
Stop the reaction by incubating at 94°C for 5 min.4.
 
Immediately begin the PCR cycle. Use 30~40 cycles with the following recommend parameters:a.
 
94°C for 30 secondsb.
 
60°C for 45 secondsc.
 
72°C for 1-3 minutes5.
 
Finish the PCR amplification with an extension at 72°C for 7 minutes.
TROUBLESHOOTINGIssue Possible Cause Suggestion
Low yield of cDNAQuality of template RNA was too low See Important Information for RNA Purity & IntegrityConcentration of RNA was too high Assay RNA concentration and dilute. Use 0.5-1µg.Reverse transcriptase inhibitor existed orreverse transcriptase was insufficientSee Important Information for RNA Purity & IntegrityReaction volume was too large Maximum volume should be 50µlLimited fulllength cDNARNA has been degradedEnsure all equipment, pipettes and gloves are RNase free.Ensure Ribonuclease Inhibitor is in the reaction.Poor reaction conditionsOptimize the quantity of reverse transcriptase, DTTconcentration (0.5-10mM) and reaction temperature(37-56°C)Inhibited by RNA secondary structure Increase reaction temperature or use a random primer

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