NxGen phi29 DNA Polymerase

Transcription

1 NxGen phi29 DNA Polymerase FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. Lucigen Corporation 2905 Parmenter St, Middleton, WI USA Toll Free: (888) (608) FAX: (608)

2 Table of Contents Product Description Product Specifications... 3 Components and Storage... 3 Reaction Setup References... 5 Technical Support Lucigen is dedicated to the success and satisfaction of our customers. Our products are tested to assure they perform as specified when used according to our recommendations. It is imperative that the reagents supplied by the user are of the highest quality. Please follow the instructions carefully and contact our technical service representatives if additional information is necessary. We encourage you to contact us with your comments regarding the performance of our products in your applications. Thank you. Lucigen Technical Support techsupport@lucigen.com Phone: (888) Product Guarantee: Lucigen guarantees that this product will perform as specified for one year from the date of shipment. Please avoid using reagents for greater than one year from receipt. 2

3 Product Description phi29 DNA Polymerase is responsible for the replication of the Bacillus Subtilis phage phi29 (1). The enzyme is a highly processive DNA polymerase (up to 70,000 base insertions per binding event) with a powerful strand displacement activity (2) and a 3 5 proofreading exonuclease function (3). Source: A recombinant E. coli strain carrying the phi29 DNA Polymerase gene from bacteriophage phi29. Product Specifications TEST SPECIFICATION Unit Concentration 10,000 U/mL Purity (SDS-PAGE) >99% SS Exonuclease Functional Endonuclease 100 U <10% converted E. coli 16S rdna Contamination 100 U <10 copies Product Designations Product Size Catalog number NxGen phi29 DNA Polymerase 2,000 Units ,000 Units Components & Storage Conditions Store all Kits and Components at -20 ºC The NxGen phi29 Polymerase package consists of the following components: Description Part Number Catalog Number Catalog Number phi29 DNA Polymerase F ,000 Units 2,000 Units x 5 10X phi29 DNA Polymerase Buffer F ml 5 x 1.5 ml 3

4 Reaction Set-Up Protocol 1: Long Incubation With Background Cleanup For each reaction: Volume, µl Component 14 Nuclease-free H 2O 2 phi29 DNA Polymerase Buffer (10X) 1 Primers* (100 µm) 1 phi29 DNA Polymerase (10,000 U/mL) 1. Add all of the above reagents, assembling reactions in a covered hood. 2. Aliquot 18 µl to PCR tubes. 3. Place tubes in a thermal cycler. 4. Incubate at 30 C for 30 minutes. 5. Place tubes on ice. a. Add 1 µl of 2.5mM dntp mixture to all tubes and mix by pipetting. b. Add 1 µl of target DNA at 10 ng/ µl to 20 ng/ µl. c. For negative control, replace target DNA with 1 µl H 2O. 6. Mix reactions by pipetting. 7. Place tubes in a thermal cycler. 8. Incubate at 30 C for 16 hours. 9. Incubate at 65 C for 10 minutes. 10. Visualize product on a 0.7% agarose gel (100 V, 40 minutes), stain with ethidium bromide. Steps 1-4 are intended to expose the buffer, primers and water to the enzyme in the absence of template. This is an optional step that will allow the enzyme s native exonuclease activity to cleanse the reaction of any contaminating DNA. Some procedures will call for a high-temperature incubation of primers with template DNA in order to enhance primer binding/annealing. Please note that if this is done, the enzyme should be added after the primer-template mixture is allowed to cool. Phi29 is not thermostable and will be denatured at high temperatures. * Primers used with phi29 are often semi-random hexamers or nonamers. They should be phosphorothioated at the two bonds on the 3 end to avoid digestion by phi29. Protocol 2: Short Incubation (No Background Cleanup) Volume, µl Component 14 Nuclease-free H 2O 2 phi29 DNA Polymerase Buffer (10X) 1 Primers (100 µm) 1 phi29 DNA Polymerase (10,000 U/mL) 1 dntps (2.5 mm) 1 Target DNA (10 20 ng/ µl) 1. Add all of the above reagents, assembling reactions in a covered hood. 2. Mix reactions by pipetting. 4

5 3. Place tubes in a thermal cycler. 4. Incubate at 30 C for 2 4 hours. 5. Incubate at 65 C for 10 minutes. 6. Visualize product on a 0.7% agarose gel (100 V, 40 minutes), stain with ethidium bromide. References 1. Blanco, L. and Salas, M. (1984) Proc. Natl. Acad. Sci. USA, 81, Blanco, L. et al. (1989) J. Biol. Chem., 264, Garmendia, C. et al. (1992) J. Biol. Chem., 267, Notice of Limited Label License, Copyright, Patents, Warranties, Disclaimers, and Trademarks A complete list of trademarks, registered trademarks, limited label license, etc. held by Lucigen Corp. can be found at 5