What would happen if you forgot to use ligase

Molecular cloning is the process used for taking recombinant DNA (referred to as an insert) and placing it into a DNA vector (i.e., plasmid) where it can be replicated and expressed. This process involves multiple steps (such as copying the DNA, cutting out the gene of interest, and pasting the gene into the DNA vector). The final step, ligation (aka the pasting step) is used to seal the insert into the vector. Ligation works by using a phosphodiester bond to connect the sugar backbone of the double-stranded DNA insert with the sugar backbone of the double-stranded DNA vector. This is typically done by using T4 DNA ligase. Índice Show Tip 1: Consider your cloning strategy Tip 2: Check the ends of your DNA inserts Tip 3: Set up ideal reaction conditions Reaction components Reaction temperatures Reaction times Tip 4: Avoid inhibitors Tip 5: Visualize your ligation reactions on a gel Tip 6: Run controls Tip 7: Check to make sure your ligase is active Summary of ligation tips T4 DNA ligase is an enzyme that helps create the formation of a phosphodiester bond between the 3'-hydroxyl end of a double-stranded DNA fragment and the 5'-phosphate end of the same or another DNA fragment (Figure 1). T4 DNA ligase can catalyze a reaction between blunt-end (no overhangs) or sticky end (3' or 5' complementary single-stranded overhangs) DNA fragments. T4 DNA ligase activity requires Mg2+ and ATP to work, and requires 5'-phosphorylation of one or both fragments. Several colonies
Cut vector (No ligase)	✓ Determines background from uncut vector ✓ Checks restriction digestion efficiency	Few colonies
Cut vector + Phosphatase + Ligase	✓ Reveals background from any recircularized vector ✓ Checks efficiency of DNA phosphatase treatment	Few colonies
Insert or water + Ligase	✓Checks for contamination of reagents, stock solutions, or pipettes	No colonies

Tip 7: Check to make sure your ligase is active

As mentioned previously, T4 DNA ligase is a temperature-sensitive ligase and is inactivated at higher temperatures. If your ligase was stored or shipped improperly, it could be denatured or lose some activity. Since your ligation reaction can fail from due to denatured or impaired ligase, you should always follow the manufacturer’s recommendations for enzyme storage conditions and usage.

If you think your ligase may have been denatured or inactivated, check the following:

The expiration date on the vial
The ligase activity by using a positive control

For example, the commercially available DNA Marker Lambda DNA/HindIII can be used as a positive control. When used in a ligation reaction with T4 DNA Ligase, the band pattern in the sample, when run on a gel, will show a single higher molecular weight band when the ligase is active. If the ligase is not active or is diminished, the original banding pattern of the Lambda DNA/HindIII marker will remain (multiple bands will show on the gel).

Summary of ligation tips

There are many reasons why ligation reactions can fail, with the most common arising from problems that occur before the addition of the T4 DNA ligase. When setting up or troubleshooting your ligation reactions, be sure to remember the tips listed below to help enable successful cloning results every time: