Cloning a PCR Fragment Into a pMAL Expression Vector (NEB #E8201)

The procedure below is for cloning a fragment produced by PCR into the pMAL-c6T vector. It is assumed that the PCR fragment is approximately 1 kb, begins with an AlwNI site, and has a SbfI overhang at the 3′ end.

  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. At the same time, run a sample of the PCR fragment to estimate its concentration.

  3. Clean up the PCR fragment using the Monarch® PCR & DNA Cleanup Kit (NEB #T1030). Then digest 0.5 μg of the PCR fragment in 20 μl of 1X CutSmart Buffer with 10 units of AlwNI and 10 units of SbfI-HF.

  4. Purify the pMAL-c6T vector backbone and the digested PCR fragment by agarose 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.

  6. Mix: 20–40 ng digested vector backbone 20 g digested insert DNA

    Add H2O to bring the volume to 5 μl

    Add 5 μl Instant Sticky-end Ligase Master Mix (2X) (NEB #M0370)

    Mix thoroughly by pipetting up and down

    Place on ice or store at –20°C for subsequent transformation.

  7. Transform 2 μl of the assembled product into competent NEBExpress cells (NEB #C2523); see transformation protocol.