Protocol for amplification of DNA using the phi29-XT WGA Kit (NEB #E1604)
- Prepare the Denaturation Solution as indicated below:
COMPONENTS VOLUME KOH (1000 mM)
100 µl
EDTA (500 mM)
5 µl
Nuclease free water
895 µl
- Denature the DNA by mixing 1-4 µl of DNA (<20 ng) or cells (≥1 cell) with 3 µl Denaturation Solution. Incubate at room
temperature for 5 minutes.
- Neutralize the denatured DNA solution by adding 3 µl of Neutralization Buffer, mix well, and place samples on ice.
- If denatured/neutralized DNA volume is less than 10 µl, bring volume to 10 µl with nuclease-free water.
- Prepare phi29-XT reaction master mix as described in the table below:
COMPONENTS SINGLE REACTION FINAL CONCENTRATION phi29-XT Reaction Buffer for WGA, 5X
4 µl
1x
Exonuclease-Resistant Random Primers, 500 µM
2 µl
50 µM
Deoxynucleotide (dNTP) Solution Mix, 10mM
2 µl
1 mM
phi29-XT DNA Polymerase for WGA, 10X
2 µl
1x
- Add 10 µl of phi29-XT reaction master mix to 10 µl of denatured/neutralized DNA. Mix well and centrifuge briefly to collect
solutions to bottom of tubes.
- Incubate in a thermocycler with the lid set at ≥ 75°C, for 1.5 hours at 42°C, followed by 65°C for 10 minutes to inactivate
the enzyme.
- The WGA products can be kept at 4°C overnight or at -20°C for long term storage.
Note: If sample cleanup is necessary, SPRI® beads are recommended, following the manufacturer’s protocol. Typically, WGA
products can be directly used in downstream applications without cleanup.