Phi29 Random Primer
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.
Ordering Info
| Cat. No | Size, µl | Price |
| R016 | 100 | $60 |
| R106 | 1000 | $410 |
| R006 | 10×1000 | $1590 |
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 µL 2X Annealing buffer* 2.5 µL Deionized water to 5 µl.
Heat 3 min at 94°C, 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
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) | ³ 1 | ³ 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 µM each) | 1.0 | 2.5 | 0.5 µM each |
| TOPOTAQ (diluted 1:5 in1X 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 94°C for 2 minutes
- Repeat the following for 30 cycles:
Denature: 94°C for 30 secondsAnneal: Primer Tm – 5°C for 30 secondsExtend: 72°C for 0.3-1 min/1 kbMaintain 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