NEBuilder Assembly of a PCR Fragment

This protocol is for cloning a fragment produced by PCR with overlapping sequences to a pMAL-c6T vector digested with AlwNI and SbfI-HF using the NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621). It is recommended to read the NEBuilder manual or use the NEBuilder Assembly Tool to create primers with the correct overlapping sequences for assembly.

  1. Digest 0.5 μg of the pMAL-c6T vector DNA in 20 μl of 1X CutSmart Buffer (supplied as a 10X stock) with 10 units of AlwNI (NEB #R0514) and 10 units of SbfI-HF (NEB #R3642) at 37°C for 1 hour. Heat inactivate the enzymes by incubating at 65°C for 10 minutes.

  2. Check for complete digestion of the pMAL-c6T vector by running 4 μl on an agarose gel.

  3. Clean up the PCR fragment using the Monarch PCR & DNA Cleanup Kit (NEB #T1030).

  4. Purify the pMAL-c6T vector backbone by gel using the Monarch DNA Gel Extraction Kit (NEB #T1020).

  5. Run a sample of the PCR insert and the vector backbone on a gel to check the concentration. The recommended DNA molar ratio is vector : insert = 1 : 2

  6. Mix: 0.015–0.1 pmol of digested vector backbone

0.03–0.2 pmol digested insert DNA

Add deionized H2O to bring the volume to 10 μl

Add 10 μl NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621)

Mix thoroughly by pipetting up and down

  1. Incubate samples in a thermocycler for 15 minutes at 50°C. Following incubation, store samples on ice or at –20°C for subsequent transformation.

  2. Transform competent NEBExpress cells (NEB #C2523) with 2 μl of the assembled product: see transformation protocol below.
  1. NEBioCalculator Thumbnail

    NEBioCalculator® - Using the ds: mass < — > moles module to plan an NEBuilder® HiFi DNA Assembly Reaction

    This tutorial describes the use of the NEBioCalculator web tool module that converts mass to, or from, moles to help plan an NEBuilder HiFi DNA Assembly reaction