Protocols for amplification of DNA using the phi29-XT RCA Kit (NEB #E1603)
Section I. phi29-XT RCA with purified circular DNA input
Input Material: Purified single or double-strand circular DNA
- Prepare reactions without enzyme as described in the table below. Mix thoroughly,
but gently, by pipetting or vortexing. Centrifuge briefly to collect solutions to
the bottom of the tube.
COMPONENTS
20 µl REACTION
FINAL CONCENTRATION
Circular DNA Template
up to 10 µl
Variable
phi29-XT Reaction Buffer, 5X
4 µl
1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM
2 µl
1 mM
Exonuclease-Resistant Random Primers 500 µM
2 µl
50 µM
Nuclease-free Water
to 18 µl
N/A
- Incubate in a thermocycler, with the lid set at 100°C, for 3 minutes at
95°C. Then allow samples to cool to room temperature.
- Place samples on ice
and add 2 µl of phi29-XT DNA Polymerase to each sample. Mix thoroughly, but gently, by pipetting or vortexing.
Centrifuge briefly to collect solutions to the bottom of tubes.
- Incubate in a thermocycler with the lid set at ≥ 75°C, for 2 hours at 42°C, followed by 10 minutes at 65°C to inactivate the DNA polymerase. The RCA products can be kept at 4°C overnight or at -20°C for long term storage.
Note: The RCA products may be viscous due to the high yield of high molecular weight DNA. A two-fold dilution with nuclease-free water is recommended before use in any downstream applications.
Note: If sample cleanup is necessary, SPRI ® beads are recommended, following the manufacturer’s protocol. Typically, RCA products can be directly used in downstream applications, such as Sanger sequencing, restriction digestion, and cell-free protein expression without cleanup.
Section II. phi29-XT RCA of plasmid DNA directly from bacterial cells
Input Material: Bacterial cells from agar plate colony, liquid media culture, or glycerol stock. This protocol is designed for mid- to high-copy number plasmids (> 10 copies per cell).
- From bacterial colony: Using a pipette tip, pick a bacterial colony from an agar plate. Resuspend it in 10 µl nuclease-free
water in a thin-walled, nuclease-free PCR tube.
Optional: Save 5 µl of bacterial suspension for liquid culture or restreak it on a new agar plate.
From liquid culture or glycerol stock: Transfer 1 µl of bacterial culture or glycerol stock to 9 µl nuclease-free water.
- To lyse cells, incubate in a thermocycler, with the lid set at
100°C, for 3 minutes
at 95°C. Then transfer samples to ice.
- Prepare reactions as described below. Mix thoroughly,
but gently, by pipetting or vortexing. Centrifuge briefly to collect solutions to the bottom of the tube.
COMPONENTS
20 µl REACTION
FINAL CONCENTRATION
Lysed cells
up to 10 µl
N/A
phi29-XT Reaction Buffer, 5X
4 µl
1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM
2 µl
1 mM
Exonuclease-Resistant Random Primers 500 µM
2 µl
50 µM
phi29-XT DNA Polymerase, 10X
2 µl
1X Nuclease-free water
to 20 µl
N/A
- Incubate in a thermocycler with the lid set at ≥ 75°C, for 2 hours at 42°C, followed by 10 minutes at 65°C to inactivate the DNA polymerase. The RCA products can be kept at 4°C overnight or at -20°C for long term storage.
Note: The RCA products may be viscous due to the high yield of high molecular weight DNA. A two-fold dilution with nuclease-free water is recommended before use in any downstream applications.
Note: If sample clean up is necessary, SPRI beads are recommended, following the manufacturer’s protocol. Typically, RCA products can be directly used in downstream applications, such as Sanger sequencing, restriction digestion, and cell-free protein expression without cleanup.
