Molecular cloning has traditionally utilized restriction enzymes to excise a fragment of interest from source DNA, and to linearize a plasmid vector while creating compatible ends. After purification of the insert and vector, both are joined with the activity of a DNA ligase, and the newly-created recombinant vector is used to transform an E. coli host for propagation of the recombinant molecule. More recently, PCR has been used to generate both the vector and insert, which can be joined using a variety of techniques, ranging from standard DNA ligation or enzymatic joining using a recombinase or topoisomerase, to homologous recombination. These newly-fashioned recombinant constructions may then be used to transform an appropriate E. coli host.
Regardless of which cloning method is chosen, the process can be made more efficient and successful by following good practices in the lab. Use NEBcloner to find the right products and protocols for each cloning step.
1. Take the time to plan your experiments
Attention to detail when planning a cloning project is essential. Ensure that your design is sound with a complete understanding of the methods being used and the sequences being generated. Pay attention to the junction sequences and the effect on reading frames of any translated sequences. Check both the vector and insert for internal restriction sites (we recommend NEBcutter®) prior to designing PCR primers that contain similar sites to those used for cloning. Verify that the antibiotic selective marker in the vector is compatible with the chosen host strain.
2. Start with clean DNA at the right concentration
Ensuring that your source DNA is free of contaminants, including nucleases and unwanted enzymatic activities, is important. Using spin-column based kits like the Monarch PCR & DNA Cleanup Kits (5 µg) (#T1030) to purify starting DNA is good practice. Completely remove solvents, such as phenol, chloroform and ethanol, prior to manipulation of the DNA. Ensure that the final elution of DNA from the spin columns is made with salt-free buffer to prevent inhibition of the downstream steps, either restriction digestion or PCR amplification. Use a sufficient amount of DNA for the technique being used. Preparative restriction digests often require between 0.2 –2.0 µg, while only single nanogram amounts are usually sufficient for DNA being used as a template for PCR.
3. Perform your restriction digests carefully
It is important to set up digestion reactions properly when you are cutting your DNA. The volume of the reaction should be compatible with the downstream step, for instance, smaller than the volume of the well of an agarose gel used to resolve the fragments. For a typical cloning reaction, this is often between 20–50 µl. The volume of restriction enzyme(s) added should be no more than 10% of the total reaction volume, to ensure that the glycerol concentration stays below 5%; this is an important consideration to minimize star activity, or unwanted cleavage.
4. Mind your ends
DNA ends prepared for cloning by restriction digest are ready for ligation without further modification, assuming the ends to be joined are compatible (have complementary overhangs or are blunt). If the ends are non-compatible, modify them using the appropriate end-modification method (e.g., use of blunting reagents, phosphatases, etc.).
DNA ends prepared by PCR for cloning may have a 3´ addition of a single adenine (A) residue as a result of amplification using Taq DNA Polymerase (NEB #M0273). High-fidelity DNA polymerases, such as Q5® (NEB #M0491), leave blunt ends. PCR using standard commercial primers produces non-phosphorylated fragments, unless the primers were 5´ phosphorylated. The PCR product may need to be kinase treated to add a 5´ phosphate prior to ligation with a dephosphorylated vector.
5. Clean up your DNA prior to vector:insert joining
For low-throughput projects, such as single gene cloning, you’ll want to clean up your digest, end modification or PCR reaction prior to proceeding. This can be achieved with gel electrophoresis or the Monarch PCR & DNA Cleanup Kits (5 µg) (#T1030). Isolating the desired DNA species and resolving it from unwanted parent vectors and/or other DNA fragments can dramatically improve your cloning results.
Confirm digested DNA on an agarose gel prior to ligation. For a single product, run a small amount of the digest, and then use the Monarch PCR & DNA Cleanup Kit (#T1030) to capture the remainder. When there are multiple fragments produced and only one is to be used, resolving the fragments on a gel and excising the desired fragment under UV light is common. Using longwave (365nm) UV light will minimize any radiation-induced DNA damage to the fragment of interest. The DNA fragment may then be recovered from the agarose slice with the Monarch DNA Gel Extraction Kit (#T1020) or β Agarase I (NEB #M0392).
6. Quantitate your isolated material
Simple quantitation methods, such as gel electrophoresis with mass standards or spectroscopic quantitation on low-input spectrophotometers (such as a NanoSpec®), ensure that the proper amount of material is used for the downstream joining reaction.
7. Follow the manufacturer’s guidelines for the joining/ligation reaction
For traditional cloning, follow the guidelines specified by the ligase supplier. If a 3:1 molar ratio of insert to vector is recommended, try this first for the best result. Using a 3:1 mass ratio is not the same thing (unless the insert and vector have the same mass). Ligation usually proceeds quickly and, unless your cloning project requires the generation of a high-complexity library that benefits from the absolute capture of every possible ligation product, long incubation times are not necessary. Follow the manufacturers’ guidelines for the joining reactions in PCR cloning and seamless cloning. If you are performing a cloning protocol for the first time, adhere to the recommended protocol for optimal results. NEB recommends using NEBioCalculator to calculate ligation ratios.
8. Use competent cells that are suited to your needs
While some labs have traditionally prepared their own competent cells “from scratch” for transformations, the levels of competence achieved rarely matches the high levels attained with commercially-available competent cells. Commercially-available competent cells save time and resources, and make cloning more reproducible.