CERTIFICATE OF ANALYSIS
悲惨世界电影AMV Rever Transcripta #EP0641 1000 u
Lot: Expiry Date:
Concentration: 20 u/µl
Supplied with: 1 ml of 5X AMV RT Buffer Store at -20°C
In total 2 vials.
Descriptionwml
AMV Rever Transcripta (RT) is a recombinant Avian Myeloblastosis Virus rever transcripta expresd li. AMV RT is a heterodimer compod of nonidentical alpha and beta subunits. It posss multiple enzymatic activities including RNA- and DNA-directed DNA polymera, DNA-RNA unwinding ability,
a quence-specific Mn2+-dependent endonuclea and RNa H. AMV RT maintains activity over a wide temperature range (45-60°C), which makes it an ideal enzyme to u in rever transcription reactions with RNAs that have a high degree of condary structure. Applications
写事的作文100字
∙First strand cDNA synthesis for RT-PCR and real-time RT-PCR, e protocol on back page.
∙Synthesis of cDNA for cloning and expression.
∙Synthesis of labeled cDNA probes.
∙Analysis of RNA by primer extension (1).
Source
Definition of Activity Unit
One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 37°C.
Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 8 mM MgCl 2, 30 mM KCl,
1 mM DTT, 0.5 mM dTTP, 0.4 MBq/ml [3
H]-dTTP, 0.4 mM polyA·oligo(dT)12-18.
Storage Buffer
The enzyme is supplied in: 200 mM potassium phosphate, 2 mM DTT, 0.2% (v/v) Triton X-100, 50% (v/v) glycerol (pH 7.2).
5X AMV RT Buffer
250 mM Tris-HCl (pH 8.5 at 25°C), 40 mM MgCl 2, 150 mM KCl, 5 mM DTT.
Inhibition and Inactivation
• Inhibitors: metal chelators, inorganic phosphate. Sodium pyrophosphate reduces AMV RT activity, however at a <4 mM concentration increas the yield of full-length cDNA (2).
• Inactivated by heating at 85°C for 5 min.
QUALITY CONTROL ASSAY DATA
Endodeoxyribonuclea Assay
No detectable conversion of covalently clod circular DNA to nicked DNA was obrved after incub
ation of 25 units of the enzyme with 1 µg of pUC19 DNA in 50 µl of reaction buffer for 16 hours at 37°C.
beefupRibonuclea Assay
0.5% of the total radioactivity was relead into trichloroacetic acid-soluble fraction after incubation of
40 units of the enzyme with 1 µg of [3
H]-RNA in 50 µl of
reaction buffer for 4 hours at 37°C.
Quality authorized by:
日语谐音Jurgita Zilinskiene
(continued on back page)
Protocol for First Strand cDNA Synthesis
The following protocol is optimized to generate first-strand cDNA for u in two-step RT-PCR.
Mix and briefly centrifuge all components after thawing, keep on ice.
1.Add into sterile, nuclea-free tube on ice in the indicated order:
total RNA
or poly(A) RNA or specific RNA 10ng-5µg
1-100 ng
0.01 pg-0.5 µg
Oligo(dT)
18 primer(#SO131) or
Random hexamer primer (#SO142) or Gene-specific primer 0.5µg (100 pmol) 0.2 µg (100 pmol) 15-20 pmol
DEPC-treated Water (#R0601) to 13µl
2.Optional: If RNA template is GC rich or is known to
contain condary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice. 3.Add the following components in the indicated order:
5X AMV RT buffer4µl
RiboLock™RNa Inhibitor
(#EO0381)
0.5 µl (20 u)
dNTP Mix, 10 mM each
(#R0191)
2 µl (1 mM final concentration)
AMV Rever Transcripta0.5µl (10u)
Total volume 20 µl
4. Mix gently and centrifuge briefly.
5.If oligo(dT)
18
primer or gene-specific primer is ud, incubate 60 min at 50°C.
If random hexamer primers are ud, incubate 10 min at 25°C followed by 60 min at 50°C.
For transcription of GC rich RNA reaction temperature
can be incread to 60°C.
6.Terminate the reaction by heating at 85°C for 5 min.
Do not heat-inactivate enzyme prior to analysis of long cDNA to avoid cleavage.
Note
astonish∙The rever transcription reaction product can be directly ud in PCR or stored at -20°C.
∙U 2 µl of the reaction mix to perform PCR in 50 µl of reaction volume.
Reference
1. Sambrook, J., Rusll, D.W., Molecular Cloning:
A Laboratory Manual, the third edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York,
没离开过 英文2001.
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2. Eun, H.-M., Enzymology Primer for Recombinant DNA
Technology, Academic Press, Inc., 1996.
PRODUCT USE LIMITATION.
This product is developed, designed and sold exclusively for rearch purpos and小学英语教学论文
painted skinin vitro u only. The product was not tested for u in diagnostics or for drug
development, nor is it suitable for administration to humans or animals.
Plea refer to for Material Safety Data Sheet of the product.
