A-Plus™ Poly(A) Polymerase Tailing Kit

A−Plus™ Poly(A) Polymeraseは、基質としてATPを使用して、RNA分子の3’末端にpoly(A)を付加します¹。A-Plus™ Poly(A) Polymerase Tailing Kitは、poly(A)を付加するのに必要な酵素やその他の試薬が入っています。

Applications

• poly(A) の付加は安定性を増加させるので、真核生物へのランスフェクション、もしくはマイクロインジェクション後の翻訳の効率を増加させます^2,3,4
• RNA分子へのpoly(A) の付加は、first-strand cDNAのためのプライマーサイトを^3'末端に作製します
• 核酸増幅もしくは遺伝子発現研究のためのploy(A)付加したRNAの合成
• RNAの3'末端のRI標識⁵
• mRNAの定量⁶

Benefits

• High purity, specificity, and activity.
• 簡便・迅速・高効率

The entire final product of an AmpliCap-MAX™ in vitro transcription capping reaction (60 mg of a 1760-base luciferase RNA) was treated with 8 Units of A-Plus™ Poly(A) Polymerase for the indicated times.

0.1 mg aliquots were run on a 1% agarose formaldehyde gel.

Unit Definition

One unit of Poly(A) Polymerase catalyzes the incorporation of 1 nanomole of AMP into acid-insoluble form in 10 minutes at 37°C in a reaction mixture consisting of 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 10 mM MgCl2, and 1 mM ATP.

Storage Buffer

50% glycerol containing 50mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton X-100.

10X A-PlusT™ Reaction Buffer

0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, and 100mMMgCl2. A separate 10mM ATP Solution is also provided.

Quality Control

A-Plus Poly(A) Polymerase is tested for polyadenylation of RNA in vitro. It is free of detectable exo- and endonuclease and RNase activity.

References

1. Gething, M et al. (1980) Nature 287, 301.
2. Drummond, DR et al. (1985) J. Cell. Biol. 100, 1148.
3. Galili, G et al. (1988) J. Biol. Chem. 263, 5764.
4. Belasco, J and Brawerman, G. (1993) Control of Messenger RNA Stability, Academic Press, San Diego, CA.
5. Lingner, J and Keller, W, (1993) Nucleic Acids Res. 21, 2917.
6. Krug, MS and Berger, SL. (1987) Methods Enzymol. 152, 262.

Kit Contents

• A-Plus™ Poly(A) Polymerase
• A-Plus™ 10X Reaction Buffer
• 10 mM ATP
• Sterile RNase-Free Water

• E. coli RNA Polymerase Core Enzyme and Holoenzyme (sigma-saturated)
• AmpliScribe T7, T3, and SP6 High Yield Transcription Kits
• Ribonuclease I, E. coli
• Ribonuclease T1 (RNase T1), Aspergillus oryzae
• T7, T3, and SP6 RNA Polymerases