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.
PDF
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 then please contact us or telephone +44 (0)1954 210 200