A-Plus™ Poly(A) Polymerase Tailing Kit

A: The A-Plus™ Poly(A) Polymerase Tailing Kit from CELLSCRIPT™ enables rapid and efficient addition of a poly(A) tail to the 3’-end of any RNA in vitro. The presence of a poly(A) tail at the 3’-end of an RNA molecule is crucial for protocols, including:

• As part of the production of mRNA in vitro.
• Increased stability and enhanced translation after transfection or microinjection of mRNA into eukaryotic cells.
• Providing a priming site for first-strand cDNA synthesis using an oligo(dT)-based primer.
• 3’-end-labeling of RNA with radioactive ATP.

A: The A-Plus™ Poly(A) Polymerase uses ATP as a substrate for template-independent addition of adenosine monophosphate to the 3’-hydroxyl termini of RNA molecules. The length of the poly(A) tail can be controlled by customizing various reaction condition parameters (e.g., reaction time, input RNA amount and amount of enzyme).

A: A standard 1X, 100 µl reaction contains 60 µg of RNA, 1X Reaction Buffer, 1 mM ATP and 8 units of A-Plus™ Poly(A) Polymerase.

A: The standard reaction conditions will generate an ~150-A tail on most average-sized RNAs in 30 minutes. Extending the incubation to 60 minutes will result in an ~200-A tail. If more precise tail lengths or longer/shorter tail lengths are desired, one can test altered parameter reactions to define reaction conditions which will result in the desired poly(A) tail length and then use those parameters for larger scale reactions.

For example, the length of the poly(A) tail can be controlled by altering the following:

• Reaction time: This is the easiest way to control poly(A) tail length. In the gel below, 1 µl samples were taken from a 1X reaction at different time points and assayed by 1% denaturing agarose gel electrophoresis. The gel shows varying tail lengths depending on the incubation time of the reaction.
  
• Input RNA amount: Maintaining all other parameters constant, the less RNA (in terms of moles of 3’ ends) in the reaction, the longer the poly(A) tail length generated.
• Amount of enzyme: Maintaining all other parameters constant, the more Poly(A) Polymerase used in the reaction, the longer the poly(A) tail generated.

A: There are several ways of stopping the tailing reaction. Depending upon the subsequent reactions the poly(A)-tailed RNA will be subjected to, the tailing reaction may be stopped by any one of the following means:

• Immediately freezing the completed reaction at -20°C or -70°C.
• Immediately proceeding to the chosen purification method.
• Removing the enzyme by phenol/chloroform-based organic extraction.
• Chelating the Mg²⁺ component of the buffer with EDTA.

Note: The reaction should not be stopped by heat denaturation since this will degrade the RNA.

A: There are several ways to purify the RNA after the poly(A)-tailing reaction:

• Salt precipitation using Ammonium acetate or Lithium chloride followed by a 70% ethanol wash.
• An RNA purification spin column.
• An RNA purification bead system such as AMPure beads (RNAClean XP).
• Phenol/Chloroform-based organic extraction followed by a salt precipitation or a size exclusion column and salt/ethanol precipitation.
• For small scale, gel purification in a polyacrylamide gel electrophoresis-based device.
• For industrial scale, tangential flow filtration followed by oligo(dT) purification.

A: For a more accurate measurement of your poly(A) tail length, CELLSCRIPT™ also offers the EZ-QC™ mRNA Poly(A) Tail Length Assay Kit.

A: The kit is guaranteed for one year from date of purchase when stored as directed at -20°C.

A: No, this enzyme will not incorporate ATP analogs.

A: No, this kit cannot be used to add poly(A) tail to DNA.