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  • Cloning a PCR Fragment Into a pMAL Expression Vector (E8200)

    The procedure below is for cloning a fragment produced by PCR into a pMAL-5 vector. It is assumed that the PCR fragment is approximately 1 kb, begins with a blunt end, and has a SbfI overhang at the 3´ end.

    1. Digest 0.5 µg of the pMAL vector DNA in 20 µl of 1X CutSmart™ Buffer (supplied as a 10X stock) with 10 units of Xmn I and 10 units of Sbf I 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 digest by running 4 µl on an agarose gel. At the same time, run a sample of the PCR fragment to estimate its concentration.
    3. Digest 0.5 µg of the PCR fragment in 20 µl of 1X CutSmart Buffer with 10 units of Sbf I.
    4. Purify the pMAL vector backbone and the cut PCR fragment by gel, or with a kit that will remove the MCS fragment of the vector and the cut off end of the PCR fragment.
    5. Run a sample of the PCR insert and the vector backbone on a gel to check the concentrations.
    6. Mix: 
      20 - 40 ng vector backbone
      20 ng insert
      Add H2O to 10 µl
      Add 10 µl 2X Quick Ligation Reaction Buffer
      Add 1 µl Quick T4 DNA Ligase (NEB #M2200)
      Incubate for 10 minutes at room temperature
    7. Mix 2 µl of the ligation reaction with 50 µl competent NEB Express and incubate on ice for 5 minutes. Heat to 42°C for 30 seconds.
    8. Add 0.2 ml SOC (or LB) and incubate at 37°C for 20 minutes. Spread on an LB plate containing 100 µg/ml ampicillin (do not plate on IPTG). Incubate overnight at 37°C.
    9. Screen for the presence of inserts in one or more of the following ways: 

      A. Perform colony PCR on a number of the transformants using appropriate primers.

      B. Prepare miniprep DNA (10). Digest with an appropriate restriction endonuclease to determine the presence and orientation of the insert (11). 

      C.
      i) Innoculate a number of transformants into 5 ml of LB amp broth and grow to 2 x 108 cells/ml (A600 of ~0.5). 

      ii) Split each sample into two 2.5 ml cultures. 

      iii) Add IPTG one of the cultures to a final concentration of 0.3 mM, for example 7.5 µl of a 0.1 M stock solution. Incubate at 37°C with good aeration for 2 hours. 

      iv) Withdraw a 0.5 ml sample from each cluture. Microcentrifuge for 1 minute, discard the supernatant and resuspend the cells in 100 µl SDS-PAGE sample buffer. 

      v) Place samples in a boiling water bath for 5 minutes. Electrophorese 10 µl of each sample on a 10% SDS-PAGE gel along with a set of protein MW standards and 15 µl of the supplied MBP2* in SDS-PAGE Sample Buffer. Stain the gel with Coomassie brilliant blue (12). 

      For pMAL-c5 constructs, an induced band should be visible at a position corresponding to the molecular weight of the fusion protein. For pMAL-p5X constructs, the induced band may or may not be visible; sometimes a Western developed with anti-MBP antibody is necessary to visualize the fusion protein. A band at or around the position of MBP5 (MW 42.5 kDa) indicates either an out of frame fusion or a severe protein degradation problem. These can usually be distinguished by performing a Western blot using the Anti-MBP Monoclonal Antibody (Appendix C); even with severe protein degradation, a full length fusion protein can usually be detected on the Western.