E.coli Poly(A) Polymerase--HYJ233
Poly(A) polymerase catalyzes the addition of AMP, converted from ATP, to the 3' end of RNA in a template-independent manner.
Components
|
Components |
100 U |
500 U |
|
E. coli Poly(A) Polymerase (5,000 U/mL) |
20 μL |
100 μL |
|
10X Poly(A) Polymerase Reaction Buffer |
1.5 mL |
1.5 mL |
|
ATP(10 mM) |
200 μL |
200 μL |
Storage
Store at -20°C.
Storage Buffer
20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 50% Glycerol, 0.1% (w/v) Triton® X-100, pH 7.5 @ 25°C
Source
Derived from an Escherichia coli strain carrying the Poly(A) polymerase gene cloned from E. coli.
Activity Definition
One unit is defined as the amount of enzyme required to catalyze the incorporation of 1 nmol of AMP into RNA in 10 minutes at 37°C in a 20 µl reaction mixture.
Applications
- Labeling RNA with ATP or cordycepin.
- Adding Poly(A) tails to RNA for cloning or affinity purification.
- Increasing mRNA stability to enhance translation efficiency in eukaryotic cells after transfection.
Inhibition and Inactivation
Inactivate at 80°C for 15 min.
Instruction
1, Add the following reaction components on ice according to the table below (example for a 20 μL reaction volume):
|
Components |
Volume |
|
10X Poly(A) Polymerase Reaction Buffer |
2μL |
|
ATP(10 mM) |
2μL |
|
E. coli Poly(A) Polymerase |
1μL |
|
RNA（1-10 μg） |
10μL |
|
ddH2O |
Up to 20μL |
2, Mix all reaction components thoroughly and briefly centrifuge to collect the solution at the bottom of the tube.
3, Incubate at 37°C for 30 minutes.
4, Heat inactivate by incubating at 85°C for 15 minutes or add EDTA to a final concentration of 10 mM.
5, RNAase inhibitor can be added to enhance RNA stability in the solution, with a 1X concentration of 1 U/μL.