Poly(A) Polymerase Tailing Kit

Components	UA070116-50 Reactions	UA070116-250 Reactions
E. coli Poly(A) Polymerase (5 U/µl)	100 μl	5 × 100 μl
10 X PAP Reaction Buffer	300 μl	5 × 300 μl
10 mM ATP	300 μl	5 × 300 μl
RNase-Free dH2O	1 ml	5 × 1 ml

1. Prepare the following reaction system on ice according to the table below:

Component	Volume	Final Concentration
10 X PAP Reaction Buffer	5 μl	1 X
RNase Inhibitor (40U/μl)	1.25 μl	1 U/μl (Optional)
E.coli Poly(A) Polymerase (5 U/μl)	2 μl	0.2 U/μl
10 mM ATP	5 μl	1 mM
RNase Free dH2O	Up to 50 μl	-

Gently pipette to mix thoroughly. 2. Poly(A) tailing reaction: Incubate at 37°C for 30–60 min. 3. Reaction termination: Immediately store at –20°C after completion, or add EDTA to a final concentration of 10 mM, or perform phenol/chloroform extraction followed by ammonium salt/ethanol precipitation of RNA.

1. Due to the involvement of RNA manipulation, strict adherence to RNA operation protocols is required to avoid RNase contamination. Related reagents and consumables must be treated with DEPC to remove RNase or ensured to be RNase-free. 2. The RNA used for the tailing reaction should be appropriately purified before use and dissolved in RNase-free dH2O. The solution should not contain EDTA or salts. 3. If it is difficult to ensure a strictly RNase-free environment, it is recommended to add an appropriate amount of RNase Inhibitor to the reaction system to enhance the stability of RNA in the solution. 4. During experimental operations, enzyme products should be kept on ice at all times and stored at -20°C immediately after the experiment.