Protocol for cloning DNA containing repeat elements (C3040)

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.
  1. 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.
  2. Mix cells and DNA gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. 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.
  5. Warm selection plates to 30°C.
  6. 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.
  7. 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.

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.