MCLAB/Poly(A) Polymerase, Yeast/PAPY30/120,000 units

Description:
Poly(A)PolymerasecatalyzesthetemplateindependentoftheadditionofAMPfromATPtothe3'-endofRNA.Poly(A)worksmorecompetentlythanE.colipoly(A)polymeraseforRNAoligonucleotide-labelingandpoly(A)tailing.Lessincubationtimeisrequiredfortheyeastenzyme.Thisenzymelabelsbothlongandshortsubstrates.Poly(A)polymerasepreferentiallylabelslongerRNA-moleculeswhereasshortRNA-moleculesarelabeledmoreefficientlybyT4RNAligase.ThereactionrequiresMn²⁺orMg²⁺,ATPassubstrates,andanyRNAcontaining3'-hydroxylterminiasprimers.LongerRNAmoleculesaresomewhatbetterprimersthanshortoligomers.Substitutionofcordycepin5'-triphosphate(3'-dATP)forATPresultsintheadditionofasingle3'-dAresiduetotheendsoftheRNA,ausefultechniqueforlabelingRNAatthe3'-end.

Application:
-Labelingthe3'-endsofRNAwithATPorcordycepin
-Poly(A)tailingofRNAforcloningoraffinitypurification
-PreparingaprimingsiteforcDNAsynthesisusingoligo-dT
-EnhancingtranslationofRNAtransferredintoeukaryoticcells

Source:
AnE.colistrainthatcarriestheclonedPoly(A)Polymerasegenefrom(Saccharomycescerevisiae).

SpecificActivity:>20,000U/mg

UnitDefinition:
Oneunitistheamountofenzymewhichincorporates1pmolAMPintoacid-insolublematerialat37°Cin1min.

5xPoly(A)PolymeraseReactionBuffer:
100mMTris-HCl,pH7.0,3.0mMMnCl₂,0.1mMEDTA,1mMDTT,500µg/mlAcetylatedBSA,50%Glycerol.

StorageBuffer:
20mMTris-HCl(pH8.0),50mMKCl,0.5mMDTT,50%Glycerol.

AssayConditions:
1xPoly(A)PolymeraseReactionBuffer,1mMrATPand500ng5'-FAMlabeledpolyA20-merRNAina20µlreaction.Afterincubationat37°Cfor10min,acidinsolubleradioactivityisdeterminedeitherbygelelectrophoresisorwithanautomatedcapillaryDNAsequencer.Inthisassay5unitsofenzymeaddapproximatley60to80adenosinestotheRNAprimer.Intheseconditons20unitsofenzymewilldepletetherATP.

HeatInactivation:65°Cfor20minutes

RecommendedStorageCondition:-20ºC

References:
1.Sippel,A.E.(1973)Eur.J.Biochem.37,31-40.
2.Edmonds,M.(1982)inTheEnzymes,3rdedition,ed.P.D.Boyer(AcademicPress,NewYork)15,217-244.
3.Gething,M.J.,Bye,J.,Skehel,J.andWaterfield,M.(1980)Nature287,301-306.
4.Sano,H.andFeix,G.(1976)Eur.J.Biochem.71,577-583.