Contributed by Ivan Delgado <email@example.com>
Ligation of a DNA fragment to a vector
T4 DNA Ligase is very unstable on ice (use as fast as posible and keep at -20oC). One unit catalyzes the exchange of 1 nmol of ATP in 20 mins at 37oC. This implies that ~1nmol of donor DNA can be ligated to an equivalent amount of acceptor DNA under these condition, in other words, use SMALL amounts of fragment and vector (otherwise there will be a lot of vector left without any fragment at the end of the reaction)
This protocol assumes that all necessary treatments to the DNA fragments (for example: DNA Fragment Isolation from LM agarose, Fill In/Chew Back of DNA overhangs, Alkaline Phosphatase removal of PO4 from DNA fragments, Kinase addition of g-phosphates to 5’-OHs) have been performed by now and that the fragments are ready to be ligated to each other.
Following restriction digestion and dephosphorylation (the two steps that can be performed in the same reaction, one after the other, without the need of purification - DNA Fragment Isolation from LM agarose-), the dephosphorylated fragment (usually vector) can be used directly without the need of a purification step (this is specially true if the vector was only linearized by digestion with a single restriction enzyme. Since there is only one fragment in the reaction, there is not need to gel purify it. In order to do this, AP has to be HEAT inactivated (Alkaline Phosphatase removal of PO4 from DNA fragments) prior to the ligation reaction
5X DNA Ligase reaction buffer (commercial) (250 mM Tris HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) polyethyene glycol (PEG)-8000. Store at -20oC. Vortex vigorously at RT when thawing)
- Prepare reaction: Mix 4 mL of 5X Ligase reaction buffer, Vector DNA (up to 0.03 nmol, up to 0.1-1 mg -see below), Insert DNA (up to 0.1 nmol, up to 0.1-1 mg -see below), and 0.1 units of Ligase (for cohesive end ligations; or 1 unit for blunt end ligations). Final reaction volume (including vector DNA, insert DNA, and dH2O) should come to 20 mL
- NOTE: the vector to insert ratio should always be at least 1:2. 1:3 is ok, but higher ratios will result in multiple inserts (if this is possible). Total DNA should not exceed 100 ng for cohesive end ligation and 1.0 mg for blunt end ligations
- Incubate at 15oC overnight*
Many different incubations can be performed. For blunt end ligations it is better to incubate at 15oC overnight. For cohesive end ligations, one hour at RT is more than enough. The bottom line is that some ligation will occur at RT regardless of the reaction (specially if enough ligase is present), so it is a matter of how much DNA was prepared and used for the ligation and the amount of the total reaction that will be used for the transformation. The more DNA the faster the reaction can be stopped and the least that needs to be used for the transformation (a portion the ligation can be taken out and used for tranformation, while the rest can be left to continue and be transformed later). Cohesive end ligations specially hold true for the above criteria. As for blunt end ligations, it is better to let them go longer or use a lot more DNA
- Although not necessary, DNA Ligase can be inactivated at 65oC for 10 minutes
- Invitrogen recomends the use of up to 5 units of DNA ligase for blunt end ligations of up to 0.18 nmol of insert with up to 0.06 nmol of vector. This has worked for me. I would discourage “rapid ligations” that have incubations of 5 minutes; the same amount of Ligase can be used for the above reactions with good results (if lower camounts have proven unsuccessful)
- Another important step, although not directly related to the ligation per se, is the competent cells used for the experiment. Commercial competent cells are extremely good and I must admit, much better than the homemade kind (Preparation of competent E. coli cells using CaCl2). Nevertheless, for most applications these homemade cells are more than enough. For important (hard) ligations, removing the uncertainty of this step by using commercial competent cells is recommended
This method is based, with permission, on an original protocol available here.