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.
- 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.
- 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.
- 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.
- Purify the pMAL-c6T vector backbone and the digested PCR fragment by agarose gel using the Monarch DNA Gel Extraction Kit (NEB #T1020).
- Run a sample of the PCR insert and the vector backbone on a gel to check the concentration.
- 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.
- Transform 2 μl of the assembled product into competent NEBExpress cells (NEB #C2523); see transformation protocol.