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.

Reagent           Quantity per reaction

Template DNA                  1 - 40 ng
Phi29 Random Hexamer Primer        1 µL
2X Annealing buffer*             2.5 µL
Deionized water                 to 5 µl
 
Heat 3 min at 94OC, cool down, then 
combine with premixed:
 
Phi29 10X buffer		   2 µL 
Phi29 DNA Polymerase             5-7 units 
dNTPs 4mM                          2 µL 
Deionized water                to 15 µL
 
Volume 				  20 µL

• Mix well by pipetting. Spin the sample briefly before placing in the thermocycler
• Incubate at 30°C for 720 minutes. 
• Inactivate polymerase at 65°C for10 min, keep at 4°C

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

PCR PROTOCOL

 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 94^oC for 2 minutes
• Repeat the following for 30 cycles:

Denature: 94°C for 30 seconds

Anneal: Primer Tm - 5°C for 30 seconds

Extend: 72°C for 0.3-1 min/1 kb

Maintain the reaction at 4°C 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 µl
ThermoFidelase 2              0.1 µL
Fimer                           1 µL
Dye Terminator Mix              2 µL
dH2O                             q.s.
Volume                         10 µL

For 96 well plate (one Fimer with 96 DNA samples), mix the following

ThermoFidelase 2 10.6 µL Fimer 106 µL Dye Terminator Mix 212 µL dH2O 106 µL

• Mix well by pipetting.
• Dispense 4.1 µL per well.
• Add 1 µL 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 µL Amplified DNA 106 µL Dye Terminator Mix 212 µL dH2O 106 µL

• Mix well by pipetting.
• Dispense 4.1 µL per well.
• Add 1 µL 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°C for 2 minutes
• Repeat the following for 200 cycles:

95°C for 5 seconds

50°C for 30 seconds

60°C for 2 minutes

• Rapid thermal ramp to 4°C and hold until ready to purify