* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Bulletin - Sigma
DNA sequencing wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Molecular evolution wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Non-coding DNA wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Biosynthesis wikipedia , lookup
Molecular cloning wikipedia , lookup
DNA supercoil wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Community fingerprinting wikipedia , lookup
SNP genotyping wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Deoxyribozyme wikipedia , lookup
ProductInformation LONG PCR CORE KIT Product No. LCOR-1 Bulletin No. MB-415 October 1998 The Polymerase Chain Reaction (PCR) is a powerful molecular biology technique now standardly used for cloning, sequencing and genome mapping. The primary enzyme used in PCR is Taq DNA polymerase. Polymerase Chain Reaction using Taq DNA polymerase is generally limited to amplifications up to 5 kb in part because Taq DNA polymerase has no 3’→5’ exonuclease or “proofreading” activity, which means periodic misincorporations are not repaired. After a misincorporation has taken place, the enzyme will either continue to incorporate nucleotides, causing a processive mistake, or a terminal event will occur and elongation will be arrested. Long and Accurate (LA) PCR combines a highly processive thermostable polymerase with a second thermostable polymerase that exhibits a 3’→5’ exonucleolytic activity. This blend increases the length of amplification products by using the proofreading polymerase to repair terminal misincorporations. This repair allows the polymerase to resume elongating the growing DNA strand. AccuTaq LA Polymerase Mix combines Sigma’s high quality Taq DNA polymerase with a small amount of a thermostable proofreading enzyme. The result is an enzyme mix that amplifies genomic targets in excess of 20 kb. Using a less complex template, such as bacterial genomic and viral DNA, amplifications of up to 20 kb and 40 kb, respectively, have been achieved. The fidelity of AccuTaq LA is 6.5 times greater than Taq DNA polymerase alone. KlenTaq LA Polymerase Mix is a blend of KlenTaq-1 DNA polymerase (a 5’-exo-minus, N-terminal deletion of Taq DNA polymerase) and a small amount of a proof-reading DNA polymerase. This blending of KlenTaq-1 and a proof-reading polymerase increases the fidelity, yield and the length of the amplified product. The usefulness of this blended enzyme is not limited to long PCR. It works extremely well for DNA amplifications 0.5-5 kb in length. KlenTaq-1 also has a broad magnesium optimum, so it is typically +2 unnecessary to optimize the Mg concentration in the reaction mixtures. It has a fidelity 4X greater than that seen in standard Taq DNA polymerase. KlenTaq LA is suitable for amplifications up to 10 kb in length. It is an excellent enzyme for amplifying cDNA templates. Sigma’s Long PCR Core Kit includes all the reagents necessary to perform long PCR on genomic DNA, bacterial genomic or viral DNA or cloned DNA templates with either Sigma’s AccuTaq LA Polymerase Mix or KlenTaq LA Polymerase Mix. Reagents • 10X Buffer for AccuTaq LA, Product 3 x 0.5 ml No. B0174 500 mM Tris-HCl, 150 mM ammonium sulfate (pH 9.3, adjusted with KOH), 25 mM MgCl2, 1% Tween 20 • 10X Buffer for AccuTaq LA minus 3 x 0.5 ml magnesium chloride, Product No. B9923 500 mM Tris-HCl, 150 mM ammonium sulfate (pH 9.3, adjusted with KOH), 1% Tween 20 C 10X Buffer for KlenTaq LA, Product 1.5 ml No. B 7673 400 mM Tricine-KOH (pH 9.2 at 25°C), 150 mM KOAc, 35 mM Mg(OAc)2, 750 :g/ml bovine serum albumin, acetylated • Magnesium Chloride, Product No. M8787 25 mM MgCl2 • Deoxynucleotide Mix, Product No. D7295 0.25 ml 10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM TTP • DMSO, Product No. D8418 • PCR Water, Product No. W1754 2 x 1.5 ml • Control Template, Product No. D8920 2.5 :g/ml lambda DNA 1.5 ml 1 ml 25 :l • Control Primer A, Product No. P1343 25 :l 10 pmol/ml Sequence: 5’-ATC ATT ATT TGA TTT CAA TTT TGT CCC ACT CCC-3' • Control Primer B, Product No. P1218 25 :l 10 pmol/ml Sequence: 5'-AGG TCG CCG CCC CGT AAC CTG TCG GAT CAC CGG AAA-3' Equipment and Reagents Required But Not Provided (Sigma Product Numbers have been given where appropriate) • • • • • • • • • AccuTaq LA DNA Polymerase Mix, Product No. D8045 or KlenTaq LA Polymerase Mix, Product No. D6290 Primers Sample DNA to be amplified Dedicated pipets PCR pipet tips 0.5 ml PCR microcentrifuge tubes, thin walled Thermocycler Mineral Oil, Product No. M 8662 (optional) Precautions and Disclaimer Sigma's Long PCR Core Kit is for laboratory use only; not for drug, household or other uses. Warning statements are included on the label or in the reagents section of this bulletin where applicable. In addition, when working with radioactively labeled nucleic acids, standard procedures for safely handling radioactive materials should be followed. Storage/Stability Store all components except magnesium chloride solution, DMSO and water below 0ºC. Store the magnesium chloride solution at 2-8°C. Store the DMSO and water at room temperature. Frozen DMSO may be melted at approx. 30ºC without any loss of performance. Note: The 10X reaction buffers for AccuTaq LA contain ammonium sulfate, which can degrade over time after repeated freeze thaw. This degradation can compromise the activity of the AccuTaq LA enzyme. If a drop in activity is observed, discard the buffer currently being used and open a new vial. Procedure For KlenTaq LA Polymerase Mix, see Sigma Technical Bulletin No. MB-365. For AccuTaq LA DNA Polymerase Mix, see Sigma Technical Bulletin No. MB-410. Technical bulletins including procedures and references are furnished with AccuTaq LA DNA Polymerase Mix and KlenTaq LA Polymerase Mix. Copies are also available from Sigma Technical Service upon request. The PCR process is covered by patents owned by Hoffman-LaRoche, Inc. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip.