DNA Polymerase for PAGE Enzyme (with 2.5 mM dNTPs)

DNA Polymerase for PAGE is purified from E. coli expressing a cloned DNA polymerase from Thermus aquaticus. The enzyme consists of a single polypeptide with a molecular weight of approximately 94 kDa. DNA Polymerase for PAGE has 5′-3′ DNA polymerase activity and 5′-3′ exonuclease activity. The extension rate is about 1-2 kb/min. This enzyme is supplied with a unique buffer compatible with PAGE, and the resulting PCR product is suitable for use in SDS-PAGE and agarose gel electrophoresis. The enzyme can amplify genomic DNA fragments up to 3 kb.

Contents:

Component	2500 U	10 kU
DNA Polymerase for PAGE	2500 U	4 × 2500 U
10X Buffer for PAGE	5 × 1.2 ml	20 × 1.2 ml
2.5 mM dNTPs	5 × 800 µl	20 × 800 µl
6X DNA Loading Buffer	1 ml	4 × 1 ml

Target	DNA Polymerase
Tested Applications	PCR
Host	Bacteria
Conjugation	Unconjugated
Biological Activity	One unit of DNA Polymerase for PAGE incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 minutes at 74 °C.
Purity	> 99% (SDS-PAGE)
Quality Control	Assayed for amplication efficiency to amplify the p53 gene from 10 ng of human genomic DNA.
Storage	Store at -20°C. Avoid repeated freeze/thaw cycles.
Shelf Life	24 months.
Buffer	DNA Polymerase for PAGE: 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 50% glycerol, stabilizers. 10X Buffer for PAGE: Contains 200 mM Tris-HCl (pH 8.3), 200 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4.
Endotoxin Level	Functional absence of double and single stranded endonuclease activity.
Concentration	5 U/µl
Availability	Shipped within 10-20 working days.
Note	THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC, THERAPEUTIC OR COSMETIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION.
Directions for use	Reaction Components: Component Volume Final Concentration Template Variable as required Forward Primer (10 µM) 1 µl 0.2 µM Reverse Primer (10 µM) 1 µl 0.2 µM 10X Buffer for PAGE 5 µl 1X 2.5 mM dNTPs 4 µl 0.2 mM DNA Polymerase 0.5-1 µl 2.5-5 U Nuclease-Free Water Variable N/A Total Volume 50 µl N/A Thermal Cycling Conditions: Number of Cycles Temperature Time per Cycle 1 cycle 94 °C 2-5 min 30-35 cycles 94 °C 30 seconds 50-60 °C 30 seconds 72 °C 1-2 kb/min 1 cycle 72 °C 5-10 min Notes: A final concentration of 2 mM MgSO4 is sufficient for amplification of most targets. For some targets, a higher Mg2+ concentration may be required. For optimal results, we recommend using a 100 mM MgSO4 stock solution to prepare a titration from 2 mM to 4 mM (final concentration) in 0.25 mM increments. 0.5 µl (2.5 U) enzyme is sufficient per 50 µl reaction volume. For better amplification, up to 1 μl (5 U) enzyme can be used.

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