Since the early 1970s, molecular cloning has used restriction enzymes to excise fragments from source DNA and to linearize plasmid vectors. After purifying the insert and vector, and ensuring compatible ends, both are joined with the help of a DNA ligase. The resulting recombinant vector is then used to transform an E. coli host into a recombinant organism, expressing the exogenous DNA.
Today, other molecular techniques are being utilized in cloning. PCR can be used to generate both the vector and insert, which can then be joined using one of a variety of techniques including:
- standard DNA ligation
- enzymatic joining using a recombinase or topoisomerase
- homologous recombination.
As with the standard procedure utilizing restriction enzymes, these recombinant constructions may then be used to transform an appropriate E. coli host.
Good laboratory practices can make cloning more efficient, no matter what method you use. Following these tips will help improve the success of your cloning experiments:
Plan your experiments
In molecular biology, the “devil” can certainly lurk in the details, so make sure your experimental design is solid, and that you understand completely the methods you’re about to use and the sequences you want to generate. You should also pay heed to junction sequences, the effect of translated sequences on reading frame and be sure to check both vector and insert for internal restriction sites (for example, using NEBcutter®). Finally you need to verify that the antibiotic selective marker in your vector is compatible with the host strain you’ve chosen.
Got clean DNA at the right concentration?
Take the steps to ensure your source DNA is free of such contaminants as nucleases and other unwanted enzymatic activities. Spin columns, to purify starting DNA, can help with this. You also need to completely remove solvents (including phenol, chloroform and ethanol) from your DNA. To keep your DNA pure, your final elution from the spin column should be made with salt-free buffer to prevent inhibition of restriction digestion or PCR amplification. Use enough DNA for your technique! Restriction digest preparations often require between 0.2–2.0 ?g of DNA, while single-nanogram amounts will suffice as a template for PCR.
Careful restriction digests
You need to ensure that the reaction volume is compatible with the downstream step. For example, if visualizing products on a gel (perhaps to gel separate inserts from vectors) the reaction volumes must be smaller than the volume of the well, to enable loading the entire sample. For a typical cloning reaction, this volume is often between 20–50 ?l. You also need to consider the volume of restriction enzyme added, which should be no more than 10% of the total reaction volume, to keep the glycerol concentration below 5%; this minimizes star activity and unwanted cleavage.
Forget the gap, mind your ends
DNA ends prepared for cloning by restriction digest are ready for ligation, assuming the ends are compatible (that is, they have complementary overhangs or are blunt). If the ends are not compatible, then they first require modification using the appropriate end-modification method, such as the use of blunting reagents or phosphatases. DNA ends prepared by PCR for cloning may have a 3´ addition of a single adenine (A) residue as a result of amplification using a Taq DNA Polymerase (e.g., Taq DNA Polymerase with Standard Taq Buffer, NEB #M0273) or have blunt ends if a high-fidelity DNA polymerase (e.g., Q5®, NEB #M0491) was used. If PCR primers used were not 5´ phosphorylated, then the fragments produced will be non-phosphorylated, and you may need to treat the PCR product with a kinase to add a 5´ phosphate prior to ligation with a dephosphorylated vector.
Clean your DNA before vector/insert joining
For single-gene cloning and other low-throughput projects, clean up your digest, end-treatment or PCR reaction before doing anything else. This can be achieved using gel electrophoresis or spin columns. This step is important as it can drastically improve your cloning by separating your DNA from unwanted parent vectors and/or other DNA fragments.
Successful digestion of your DNA should be confirmed on an agarose gel prior to ligation. For a digest where there is only a single product, you can run a small amount on the gel (to check for digestion), and then use a spin column to capture the remainder. However, if you’ve produced multiple fragments but only intend to use one, you need to resolve the fragments on a gel and excise the desired fragment under UV light. Long-wave (365 nm) UV light is preferable, as this will minimize any radiation-induced DNA damage. Once excised, the DNA can then be recovered from the agarose slice using either a gel extraction kit or ? agarase I (NEB #M0392).
Make it Count – Quantitate!
Ensure that the right amount of DNA is used in the downstream joining reaction by quantifying your DNA using methods such as gel electrophoresis with mass standards or spectroscopic quantitation on low-input spectrophotometers.
Follow manufacturer’s guidelines for the joining reaction
For the best results with traditional cloning ensure you follow the guidelines for your chosen ligase. For example if a 3:1 molar ratio of insert to vector is recommended, try this first, but be aware that this is not the same as using a 3:1 mass ratio (unless the insert and vector have the same mass). Ligation usually proceeds quickly and, in most circumstances, long incubation times are not necessary. If, however, your cloning project benefits from the capture of every possible ligation product, longer incubation times may be beneficial, for example, in the generation of a high-complexity library. As with ligase reactions, you should follow the manufacturers’ guidelines for joining reactions in PCR cloning and seamless cloning.
Use the right competent cells – consider commercial sources
Many labs have traditionally prepared their own competent cells. However the competence levels from these “home brews” rarely match those attained from commercially-available competent cells. The use of commercially-available competent cells is recommended in order to save time and resources, as well as to ensure reproducibility of cloning.