ORDERING INFO
Cat. No
Size, オl
R015
100
R105
1000
R005
10x1000
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
LINKSphi 29 DNA polymerase
(1989) JBC 264, 8935-8940 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
DESCRIPTIONPhi29 Random Pentamer 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 Pentamer Primer is optimized for amplification with Phi29 DNA
Polymerase at room temperature.
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 Pentamer Primer 1 オL
2X Annealing buffer* 2.5 オL
Deionized water to 5 オl
Heat 3 min at 94OC, cool down, then
combine with premixed:
Phi29 10X buffer 2 オL
Phi29 DNA Polymerase 5-7 units
dNTPs 4mM 2 オL
Deionized water to 15 オL
Volume 20 オL
- Mix well by pipetting. Spin the sample briefly before placing in
the thermocycler
- Incubate at 25ーC for 720 minutes.
- Inactivate
polymerase at 65ーC for10 min,
keep at 4ー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)
ウ
1
ウ
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 オM each)
1.0
2.5
0.5 オM 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ーC for 30 seconds
- Anneal: Primer Tm - 5ーC for 30 seconds
- Extend: 72ーC for 0.3-1 min/1 kb
- Maintain the reaction at 4ー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 オl
ThermoFidelase 2 0.1 オL
Fimer 1 オL
Dye Terminator Mix 2 オL
dH2O q.s.
Volume 10 オL
- For 96 well plate (one Fimer with 96 DNA samples), mix the following
ThermoFidelase 2 10.6 オL
Fimer 106 オL
Dye Terminator Mix 212 オL
dH2O 106 オL
- Mix well by pipetting.
- Dispense 4.1 オL per well.
- Add 1 オL 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 オL
Amplified DNA 106 オL
Dye Terminator Mix 212 オL
dH2O 106 オL
- Mix well by pipetting.
- Dispense 4.1 オL per well.
- Add 1 オL 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ーC for 2 minutes
- Repeat the following for 200 cycles:
- 95ーC for 5 seconds
- 50ーC for 30 seconds
- 60ーC for 2 minutes
- Rapid thermal ramp to 4ーC 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.