A-Plus™ Poly(A) Polymerase Tailing Kit

Create variable 3’ poly(A) tail lengths from 50 to >300 A’s through post-transcriptional tailing.
Generate longer poly(A) tails than lengths possible using template-encoded tails.
Synthesis of long (300+ A) tail lengths is possible for increased mRNA stability and translation.
Simplified workflow with direct input of capped RNA from ScriptCap™ reactions with no cleanup required.

Product is for research use only (RUO)

Product Description:

The A-Plus™ Poly(A) Polymerase Tailing Kit uses ATP as a substrate for template-independent post-transcriptional addition of 3’-poly(A) tails to RNA. The standard protocol produces a poly(A) tail length of ~150 A’s on 60 µg of RNA in just one hour. However, the flexible protocol allows users to define their desired poly(A) tail length from 50–300+ A’s using straightforward modifications. In this way, poly(A) tail lengths can be generated much longer than is possible using a template-encoded tail. Completed ScriptCap™ Cap 1 Capping System capping reactions can be added directly to the A-Plus™ poly(A) tailing reaction without cleanup for seamless synthesis of fully 5’-capped and 3’-tailed mRNA.

Polyadenylation increases the stability of RNA in eukaryotic cells and enhances its ability to be translated after transfection or microinjection. 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-label or quantify mRNA.

Performance Data:

Poly(A) Tail Length

The standard protocol generates poly(A) tails approximately 150 b long. Assuming all other reaction parameters remain constant, poly(A) tail length increases with the following parameter changes:

Increasing units of A-Plus™ Poly(A) Polymerase
Increasing incubation time
Increasing enzyme to substrate ratio
Decreasing total reaction volume

To find the best reaction conditions for the desired poly(A)-tail length, set-up several test reactions covering a range of the changing parameter (Figure 1).

Figure 1. Different poly(A) tail lengths generated using the A-Plus™ Poly(A) Polymerase Tailing Kit. The standard reaction results in a poly(A) tail length of approximately 150 bases, but longer tail lengths can be achieved by adjusting enzyme concentration, incubation time, RNA input, and reaction volume.

Materials Supplied:

Important 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), 0.1 mM EDTA and 0.1% Triton® X-100	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
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

Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.

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Troubleshooting Guide:

Symptom	Solution
Poly(A) tails are longer than expected	Decrease the time of incubation of the reaction.
	Decrease the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
	Increase the concentration of substrate RNA in the reaction by using more RNA, or decreasing the total reaction volume.
Poly(A) tails are shorter than expected	Increase the time of incubation of the reaction.
	Increase the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
	Decrease the concentration of substrate RNA in the reaction by using less RNA, or increasing the total reaction volume.
No poly(A) tails are observed	The RNA 3' end may be buried in secondary structure. Perform the RNA heat-denaturation step.
	The RNA substrate is contaminated. Repurify the RNA substrate.
	Enzyme is inactive. Store only at –20°C. Keep on ice when not in the freezer.
	ATP is hydrolyzed. Do not expose to elevated temperatures.