For best results, assemble insert fragment and vector fragment using the NEBuilder® HiFi DNA Assembly Master Mix (NEB# E2621). PCR amplification of either insert or vector DNA should be carried out using a high fidelity DNA polymerase such as Q5® High Fidelity DNA Polymerase (NEB# M0491 or M0493). If using an assembly approach, use PCR primer design software such as NEBuilder.
Note: For best clone stability, incubate plates and liquid
cultures at 30°C and prepare plasmid DNA from fresh transformants (from
plates not greater than 3 days old). Store unstable clones as plasmid
DNA, rather than cell-based glycerol stocks.
- Add 2 µl of the 20 µl NEBuilder HiFi Assembly reaction to NEB Stable competent cells. Alternatively, add 2 µl of a 20 µl ligation reaction to NEB Stable competent cells.
- Mix cells and DNA gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. Place on ice for 5 minutes. Do not mix. Pipette 950 µl of room temperature NEB® 10-beta/Stable Outgrowth Medium into the mixture. Incubate the cells with shaking at 30°C for 60 minutes. Shake the tube horizontally at 250rpm or rotate.
- Warm selection plates to 30°C.
- Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µl of cells or diluted cells onto a selection plate and incubate at 30°C for 24 hours.
- Analyze at least 6 clones by colony PCR or directly grow individual clones in liquid culture at 30°C or 37°C to isolate plasmid DNA.