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.
- 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.
- 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.
- Digest 0.5 µg of the PCR fragment in 20 µl of 1X CutSmart Buffer with 10 units of Sbf I.
- 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.
- Run a sample of the PCR insert and the vector backbone on a gel to check the concentrations.
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
- 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.
- 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.
- 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).
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.