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.