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In vitro Transcription

In vitro Transcription: In Vitro Transcription

A-Plus Poly(A) Polymerase Tailing Kit

For post-transcriptional poly(A) tail addition to RNAs. Allows tailing of mRNAs in vitro, to lengths equivalent to or greater than those found in nature. This cannot be accomplished with co-transcriptional tailing methods.

CellScript

Catalogue No.DescriptionPack SizePriceQty
C-PAP5104HA-Plus™ Poly(A) Polymerase Tailing Kit50 rxns £163.00 Quantity Add to Order

A-Plus Poly(A) Polymerase Tailing Kit

For post-transcriptional poly(A) tail addition to RNAs. Allows tailing of mRNAs in vitro, to lengths equivalent to or greater than those found in nature. This cannot be accomplished with co-transcriptional tailing methods.

CellScript

The A-Plus™ Poly(A) Polymerase Tailing Kit uses ATP as a substrate for template-independent addition
of adenosine monophosphates to the 3'-hydroxyl termini of RNA. The standard protocol produces a
poly(A)-tail length of ~150 b on 60 μg of RNA. Polyadenylation increases the stability of RNA in
eukaryotic cells and enhances its ability to be translated after transfection or microinjection.1-3 A Poly(A)
tail is useful to provide a priming site for first-strand cDNA synthesis in certain applications, and can be
used to end-label4 or quantify5 mRNA.

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The nuts and bolts of translational efficiency and the "closed-loop" model

 

MATERIALS
Materials Supplied
Store at –20°C in a freezer without a defrost cycle. Do not store at –70°C.

A-Plus™ Poly(A) Polymerase Tailing Kit Contents (50 reactions)
Kit Component Volume
A-Plus™ Poly(A) Polymerase, 4 U/μl
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM
dithiothreitol (DTT) and 0.1 mM EDTA.
100 μl
10X A-Plus™ Poly(A) Tailing Buffer
0.5 M Tris-HCl, pH 8.0, 2.5 M NaCl and 100 mM MgCl2.
500 μl
10 mM ATP 500 μl
RNase-Free Water 2 x 1.4 ml

 

Materials Required, but not Supplied
• Materials or kits for purification of the RNA product. (For suggestions, see RNA Purification, page 4)
• RNase-free TE Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA)
• Optional: TE saturated phenol/chloroform, 0.5-1 M EDTA, ScriptGuard™ RNase Inhibitor


SPECIFICATIONS
Unit Definition
One unit of A-Plus Poly(A) Polymerase converts 1 nmole of ATP into acid-insoluble material in 10
minutes at 37oC under standard assay reaction conditions.


Functional Testing
The A-Plus Poly(A) Polymerase Tailing Kit is functionally tested in 1X A-Plus Poly(A) Tailing Buffer
with 1 mM ATP, a 1.4 kb RNA transcript and varying amounts of A-Plus Poly(A) Polymerase.
Contaminating Activity Assays
All components of the A-Plus Poly(A) Polymerase Tailing Kit are free of detectable RNase and DNase
activity.

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

A-Plus Poly(A) Polymerase Tailing Kit

For post-transcriptional poly(A) tail addition to RNAs. Allows tailing of mRNAs in vitro, to lengths equivalent to or greater than those found in nature. This cannot be accomplished with co-transcriptional tailing methods.

CellScript

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Please note: all protocols are the responsibility of the product supplier

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

A-Plus Poly(A) Polymerase Tailing Kit

For post-transcriptional poly(A) tail addition to RNAs. Allows tailing of mRNAs in vitro, to lengths equivalent to or greater than those found in nature. This cannot be accomplished with co-transcriptional tailing methods.

CellScript

REFERENCES
1. Drummond, D.R. et al., (1985) J. Cell. Biol. 100, 1148.
2. Galili, G. et al., (1988) J. Biol. Chem. 263, 5764.
3. Belasco, J. and Brawerman, G. (1993) Control of Messenger RNA Stability, Academic Press, San
Diego, CA.
4. Lingner, J. and Keller, W. (1993) Nucleic Acids Res. 21, 2917.
5. Krug, M.S. and Berger, S.L. (1987) Methods Enzymol. 152, 262.
6. Sambrook, J et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), New York, Cold Spring
Harbor Laboratory Press.

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200