Preparation of insert and vector DNA
Preparation of DNA fragments
To ligate a DNA fragment into a plasmid vector you have first of all to prepare your fragment and your vector in a way that your fragment can be inserted into the vector.
For this fragment (also called insert) and vector must have compatible ends after digestion.
If you use for the digestion of your insert and vector the same enzyme, producing cohesive ends the ends of your DNA fragments will be compatible anyway. All blunt ends are compatible with each other. However there are some restriction enzymes that have different recognition sites, but compatible cohesive. This creates a possibility to clone insert digested with one enzyme into a vector digested with a different one. You can check catalogue for such compatible enzymes, one example is a pair SalI and XhoI.
The most critical component of the reaction is your vector. It has to be digested as good as it possible, therefore I recommend to set a reaction in a relatively big volume (50-100 µl). Use excess of restriction enzyme and prolong incubation (however don’t forget about star activity!). Although there are always debates regarding the necessity of dephosphorylation of the vector, I recommend dephosphorylating the vector in any case. Even if you use two different enzymes for digestion, additional dephosphorylation will reduce the background coming from religation of the empty vector. Make a fresh agarose gel and run it slower than usual. Digested vector has to be very good separated from the non-digested one.
Insert preparation is less critical, since it is usually easier to separate it from the source plasmid. One more tip here. If your insert has the same size as the remaining part of the source vector (so you can’t separate them on a gel) find enzymes that cut your vector, leaving the insert intact.
To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction enzymes that create compatible ends. At least one of the enzymes used should be a sticky end cutter to ensure that the insert is incorporated in the right orientation.
It will save you a lot of time when you could carry out the two digestions simultaneously (double digestion). Not all restriction enzymes work equally well in all commercially available buffers and, therefore, it is worthwhile to checkwhich enzymes are compatible and in which buffer. To ensure efficient digestion the two recognition sites should be more than 10 base pairs apart. If one of the enzymes is a poor cutter or if the sites are separated 10 base pairs or less, the digestions should be performed sequentially. The first digest should be done with the enzyme that is the poorest cutter and the second enzyme added after digestion has been verified by running a sample of the reaction mix on an agarose gel.
Vector Preparation
· Digestion of vector DNA using (preferably) two restriction enzymes.
· Dephosphorylation of the ends using calf intestine or shrimp alkaline phosphatase. This reduces the background of non-recombinants due to self-ligation of the vector (especially when a single site was used for cloning).
· Purification of the digested vector by agarose electrophoresis to remove residual nicked and supercoiled vector DNA and the small piece of DNA that was cut out by the digestions. This usually reduces strongly the background of non-recombinants due to the very efficient transformation of undigested vector.
Insert Preparation
· Digestion of insert DNA using (preferably) two restriction enzymes.
· Purification of the digested insert. Purification should be carried out by agarose gel electrophoresis when the insert is subcloned into a vector from a vector with the same selective marker or PCR amplified from a vector with the same selective marker. Otherwise, it can be purified using a commercial kit.
Sources:
Content:
https://www.methods.info/Methods/RNA_DNA/ligation_simple.html