PRODUCT INFORMATIONExonuclease IIIExo IIILot Expiry Date _Concentr - PDF document

PRODUCT INFORMATIONExonuclease III(Exo III)Lot: Expiry Date: _Concentration: Supplied with: ml of 10X Reaction Buffer Store at 20°C Description Exonuclease III (ExoIII) exhibits four different ApplicationsCreation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease (2, 3), see protocol on back pageGeneration of a singlestranded template for dideoxy sequencing of DNA (4).Sitedirected mutagenesis (5).Cloning of PCR products (6).Preparation of strandspecific probes.SourceE.colicells carrying a cloned E.coli xthgene.Molecular Weight31 kDa monomer.Definition of Activity Unit One unit of the enzyme atalyzes the release of 1nmol of acid soluble reaction products from E.colii3H]-DNA in min at 37°C.Enzyme activity is assayed in the following mixture: mM TrisHCl (pH 8.0), 5mM MgClmM DTT and sonicatedE.colii3H]-DNA. Storage ufferThe enzyme is supplied in: 50mM TrisHCl (pH 8.0), mM KCl, 1DTT and 50% (v/v) glycerol.10X ReactionBuffermM TrisHCl (pH 8.0 at 30°C), 6.6MgClInhibition and InactivationInhibitors: metal chelators, pchloromercuri benzoate 90% inhibitory at 0.1 mM) (7).Inactivated by heating at 70°C for 10min.NoteThe rate of DNA digestion by Exodepends upon temperature, salt concentration and the molar ratio of DNA to enzyme in a reaction

mixture (4, ). Optimal reaction conditions should be determined experimentally.Activity in Thermo ScientificREase Buffers, %(in comparison to activity in assay buffer) Buffers Activity, % Thermo Scientific FastDigest, FastDigest ® Green, O, R, X Thermo Scientific Tango, BamHI, EcoRI G, ango B , Ecl136II, KpnI , PacI, SacI 02525-50 100 CERTIFICATE OF ANALYSISEndodeoxyribonuclease AssayNo conversion of covalently closed circular DNA to nicked DNA was detectedafter incubation of 25 units of Exonuclease IIIwith 1g of pUC19DNA for4 hours at 37°C.Quality authorized by:Jurgita Zilinskiene(continued on back page) Protocol for Generation of Unidirectional Deletions in DNA Fragments (for 25 time points)Digest 5µg of DNA with a pair of restriction enzymes; one which generates a blunt or overhanging end adjacent to the target sequence, and another which produces a resistant 4 base overhang close to the priming site.Extract the reaction mixture with 1 volume of phenol:chloroform (1:1) and then with 1 volume of chloroform:isoamyl alcohol (24:1). Precipitate DNA by adding 0.1 volume of NaCl/glycogen solution (1.1NaCl, 0.25mg/ml glycogen) and 2 volumes of 95% ethanol. Mix and centrifuge at 10,000rpm for minutes. Wash the pellet with 1ml of 70% ethanol.Dry the pellet.Dissol

veDNA in 50l of 1X ExoIII reaction buffer.Preparemixture (S1 Nuclease mix) by adding the following: 5X R eaction B uffer for S1 Nuclease* 40 µl S1 Nuclease (#EN0321) 0.5 µl (50 u) Water, nuclease - free (#R0581) to 200 µl mM sodium acetate (pH 4.5 at 25°C), 1.5M NaCl and mM ZnSOAdd 7.5µl aliquots of the S1 Nuclease mix into each of 25 numbered tubes, place on ice.Selectincubation temperature and time as follows: Temperature, °C Digestionrate, bp/min Mononucleotides are released at basedependent rates in the order: C�A=T�G. DNA termini of different are degraded at different rates, so these guidelines should only be used as an approximation.Warm the DNA solution to the digestion temperature. Add 500 unitsExoIII and mix rapidly. Remove 2µl aliquots at the chosen time intervals and add to the tubes containing the cold S1Nuclease mix.After all samples have been taken, move the tubes to room temperature for 30minutes. Add 1µl S1 "STOP" solution(300Tris base, 50EDTA) and heat at 70°C for 10minutes to inactivateS1 Nuclease.Usel of each sample for gelelectrophoresis. References 1. Rogers, S.G. and Weiss, B., Exonuclease III of Escherichia coli12, an AP endonuclease, Methods Enzymol., 65, 2012. Hoheisel, J.D., On the Activities of Escheri

chia coliExonuclease III, Anal.Biochem., 209, 238. Henikoff, S., Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing, Gene, . Guo, LiHe., Wu, R., New rapid methods for DNA sequencing based on exonuclease III digestion followed by repair synthesis, Nucleic Acids Res., 10, 2065. Vandeyar, M.A., A simple and rapid method for the selection of oligodeoxynucleotidedirected mutants, Gene, 65, 129133, 1988.. Li, Ch., Evans, R.M., Ligation independed cloning irrespective of restriction site compatibility, Nucleic Acids Res., 25, 41654166, 1997.Eun, H.M. Enzymology Primer for RecombinantTechnology, Academic Press. Inc., 1996.8. Tomb, J.F., Barcak, G.J., Regulating the 3'5' activity of exonuclease III by varying the sodium chloride concentration, BioTechniques, 7, 932 PRODUCT USE LIMITATION This product is developed, designed and sold exclusively for research purposes and in vitro use only.The product was not tested for use in diagnostics or for drug deveopment, nor is it suitable for administration to humans or anima Please refer to www.thermoscientific.com/onebio for Material Safety Data Sheet of the product. Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiari