Low temperatures generally reduce ligase activity, whereas too high temperatures may reduce cloning efficiencies by melting annealed DNA overhangs and increase overall molecular motion in the ligation reaction.
Several procedures have been described to increase the efficiency ligation reactions, including the addition of condensing agents as polyethylene glycol 1 or hexamminedicobalt chloride 2.
These approaches induce macromolecular crowding, and thus serve to mimic higher DNA concentrations in the ligation reaction. Other approaches seek to increase the efficiency of molecular cloning procedures by omitting the ligation step by generating long single-stranded DNA overhangs that can be annealed and transformed directly into an appropriate Escherichia coli host 3. We have devised a simple procedure in which high enzyme activity and DNA annealing is balanced by constant temperature cycling.
All enzymes were purchased from Amersham. The purified fragments were added in equimolar amounts. Competent XL1-Blue E. The number of colonies appearing per transformation is shown in Table 1. We have tested the kinetics of temperature-cycle ligations over 12—16 h, and found them to be similar to those of ligations at constant temperatures resulting in a linear increase with time in the number of colonies appearing after transformation data not shown.
Furthermore, temperature-cycle ligation may be used together with hexamminedicobalt chloride in blunt-end ligations to give the combined effect.
Temperature-cycle ligation is routinely used in our laboratory, and, we believe the technique to be broadly applicable in all protocols involving molecular cloning, with special relevance to difficult clonings and library construction.
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Sign In. Advanced Search. Search Menu. Article Navigation. Gently mix the reaction and centrifuge briefly. Incubate the reactions. Heat-inactivate the ligation reactions.
Why is heat inactivating T4 DNA ligase necessary? Ligation reactions fail due to these following possible reasons: 1.
The presence of ligase inhibitors in the reactions To prevent contaminations of ligase inhibitors in the reactions, such as high salts or EDTA, make sure you purify your DNA insert and plasmid. To find out how much insert DNA to add in a ligation reaction if you want the ratio to be , use the formula below: 3. ATP concentration in the buffer is too low If the ATP concentration in the ligation reaction is too low, the ligation will fail. The inactive enzyme The ligation reaction might also fail due to the inactive enzyme.
Set up also a negative control with no addition of ligase. If your ligase is still active, you should observe a distinct band with a higher molecular weight for the reaction with the ligase compared to the one without the ligase.
T References Johnson, A. Related Articles. This particul Molecular Cloning: A Detailed Introduction. Molecular cloning is a primary procedure in contemporary biosciences. This involves introducing a sp Since the early s, restriction enzymes have become an important part of cloning and many other a In molecular biology, agarose gel electrophoresis is a common method used for many applications, suc Gold Biotechnology U.
Registration No 3,, and Goldbio U. Registration No 3,, are registered trademarks of Gold Biotechnology, Inc. We use cookies and other tools on this site. Our DNA ligation kit for long fragments is a powerful tool for cloning long DNA fragments spanning 2 kb to over 10 kb in length. It is especially well-suited for BAC library construction. In addition, this kit also enables fast and easy ligation of smaller-sized DNA inserts. It is especially well-suited for the construction of BAC libraries.
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