Phi29 Random Hexamer Primer

ORDERING INFO
Cat. No Size, 痞
R016 100
R106 1000
R006 10x1000
RC06 100000

Contact Fidelity Systems if you need biotin, amino group, dyes or other modifiers attached to your Random Primer.


CONTACT INFO
301-527-0804 (tel)
301-527-8250 (fax)
fsi1@fidelitysystems.com



LINKS

phi 29 DNA polymerase
(1989) JBC 264, 8935-8940

Cell-free cloning using phi29 DNA polymerase and exonuclease-resistant hexamer mix
(2005) PNAS 102, 17332-17336


Highly sensitive sequencing from genomic DNA
(2002) PNAS 99, 4644-4649

No Error BAC sequencing

Bacterial Artificial Chromosomes Vol. 1, Chapters 21 and 16



FOR RESEARCH USE ONLY
PRODUCT DESCRIPTION
Phi29 Random Hexamer Primer is ready-to-use in strand displacement amplification of genomic DNA, plasmids, BACs, fosmids or phages.

Phi29 Primer contains modified nucleotides that are resistant to 3'-5' exonuclease activity. We have developed proprietary purification procedure to ensure removal of DNA polymerase inhibitors. The concentration of Phi29 Random Hexamer Primer is optimized for amplification with Phi29 DNA Polymerase.

A method for the cell-free cloning of single DNA molecules without propagation in bacteria using phi29 DNA polymerase and our exonuclease-resistant Phi29 Random Hexamer Primer mix was developed by Clyde A. Hutchison, III, Hamilton O. Smith, Cynthia Pfannkoch, and J. Craig Venter (Synthetic Biology Group, The J. Craig Venter Institute) and published in PNAS.

MATERIALS SUPPLIED BY THE USER
Template DNA, Phi29 DNA Polymerase with Buffer and dNTPs,  Deionized H2O.

DNA AMPLIFICATION
Reagent           Quantity per reaction

Template DNA 1 - 40 ng Phi29 Random Hexamer Primer 1 無 2X Annealing buffer* 2.5 無 Deionized water to 5 痞

 

Heat 3 min at 94OC, cool down, then combine with premixed:

 

Phi29 10X buffer 2 無  Phi29 DNA Polymerase 5-7 units  dNTPs 4mM 2 無  Deionized water to 15 無

 

Volume 20 無
 
  • Mix well by pipetting. Spin the sample briefly before placing in the thermocycler
  • Incubate at 30蚓 for 720 minutes. 
  • Inactivate polymerase at 65C for10 min, keep at 4C
To avoid extra DNA purification steps and ensure high sensitivity, you may use the following protocols for PCR and sequencing.

*2X Annealing buffer: 80 mM Tris-HCL, pH 8.0; 20 mM MgCl2

PCR PROTOCOL

Components

Volume ml

Final Conc

Amplified DNA (10 pg - 200 ng)

1

1

As required

2X Amplification Buffer

    with 6 mM MgCl2

10

25

1X; 3 mM MgCl2

      dNTP mixture

 (10 mM each dNTP)

1.0

2.5

0.5 mM each

Primer mixture (10 然 each)

1.0

2.5

0.5 然 each

TOPOTAQ  (diluted 1:5 in

1X TOPOTAQ  Dilution buffer )

1.0

2.0

1-2 U

Distilled water

to 20

to 50

 

 Mix content of the tubes and overlay with mineral or silicone oil if necessary

Cap the tubes and centrifuge briefly to collect the contents

Use the following cycling conditions:
  • Heat the plate at 94oC for 2 minutes
  • Repeat the following for 30 cycles:
Denature: 94蚓 for 30 seconds
Anneal: Primer Tm - 5蚓 for 30 seconds
Extend: 72蚓 for 0.3-1 min/1 kb
Maintain the reaction at 4蚓 after cycling


CYCLE SEQUENCING

For sequencing of less than 96 samples, use the following table for calculation of the amount of each component

Reagent           Quantity per reaction
Amplified DNA                   1 痞
ThermoFidelase 2              0.1 無
Fimer                           1 無
Dye Terminator Mix              2 無
dH2O                             q.s.
Volume                         10 無

For 96 well plate (one Fimer with 96 DNA samples), mix the following
ThermoFidelase 2             10.6 無
Fimer                         106 無
Dye Terminator Mix            212 無
dH2O                          106 無
  • Mix well by pipetting.
  • Dispense 4.1 無 per well.
  • Add 1 無 of Amplified DNA in each well. Seal the plate.
  • Spin the plate briefly before placing in the thermocycler.
For 96 well plate (96 Fimers with one DNA sample), mix the following
ThermoFidelase 2             10.6 無
Amplified DNA                 106 無
Dye Terminator Mix            212 無
dH2O                          106 無
  • Mix well by pipetting.
  • Dispense 4.1 無 per well.
  • Add 1 無 of Fimer in each well. Seal the plate.
  • Spin the plate briefly before placing in the thermocycler.
Use the following cycling conditions:
  • Heat the plate at 95蚓 for 2 minutes
  • Repeat the following for 200 cycles:
95蚓 for 5 seconds
50蚓 for 30 seconds
60蚓 for 2 minutes
  • Rapid thermal ramp to 4蚓 and hold until ready to purify

Covered by U.S. Patents 5,427,928, 5,656,463, 5,902,879, 6,548,251. Patents pending. Fidelase, ThermoFidelase, D-Strap and Fimer are trademarks of Fidelity Systems.
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