NxGen® phi29 DNA
Polymerase
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA
Toll Free: (888) 575-9695 | (608) 831-9011 | FAX: (608) 831-9012
[email protected] www.lucigen.com
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NxGen® phi29 DNA Polymerase
Table of Contents
Product Description... .............................................................................................................................3
Product Specifications… ........................................................................................................................3
Components and Storage…...................................................................................................................3
Reaction Setup...... .................................................................................................................................4
References… .........................................................................................................................................5
Technical Support
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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.
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Email: [email protected]
Phone: (888) 575-9695
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.
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NxGen® phi29 DNA Polymerase
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
Unit Concentration
Purity (SDS-PAGE)
SS Exonuclease
Endonuclease
E. coli 16S rDNA Contamination
SPECIFICATION
10,000 U/mL
>99%
Functional
100 U <10% converted
100 U <10 copies
Product Designations
Product
Size
Catalog number
2,000 Units
30221-1
10,000 Units
30221-2
NxGen® phi29 DNA Polymerase
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
30221-1
Catalog Number
30221-2
phi29 DNA Polymerase
F83900-1
2,000 Units
2,000 Units x 5
10X phi29 DNA Polymerase Buffer
F88901-1
1.5 mL
5 x 1.5 mL
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NxGen® phi29 DNA Polymerase
Reaction Set-Up
Protocol 1: Long Incubation With Background Cleanup
For each reaction:
Volume, µL Component
14
Nuclease-free H 2 O
2
phi29 DNA Polymerase Buffer (10X)
1
Primers* (100 µM)
1
phi29 DNA Polymerase (10,000 U/mL)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Add all of the above reagents, assembling reactions in a covered hood.
Aliquot 18 µL to PCR tubes.
Place tubes in a thermal cycler.
Incubate at 30 °C for 30 minutes.
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 2 O.
Mix reactions by pipetting.
Place tubes in a thermal cycler.
Incubate at 30 °C for 16 hours.
Incubate at 65 °C for 10 minutes.
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
14
2
1
1
1
1
Component
Nuclease-free H 2 O
phi29 DNA Polymerase Buffer (10X)
Primers (100 µM)
phi29 DNA Polymerase (10,000 U/mL)
dNTPs (2.5 mM)
Target DNA (10 – 20 ng/ µL)
1. Add all of the above reagents, assembling reactions in a covered hood.
2. Mix reactions by pipetting.
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NxGen® phi29 DNA Polymerase
3.
4.
5.
6.
Place tubes in a thermal cycler.
Incubate at 30 °C for 2 – 4 hours.
Incubate at 65 °C for 10 minutes.
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, 5325-5329.
2. Blanco, L. et al. (1989) J. Biol. Chem., 264, 8935-8940.
3. Garmendia, C. et al. (1992) J. Biol. Chem., 267, 2594-2599.
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