Ligation Calculator
Determines the optimal insert amount for a cloning reaction based on vector and insert properties.
Refer to the text below the tool for more information about the formula used and about molar ratios for cohesive or blunt end ligations.
During the ligation rection, a recombinant plasmid is created from covalently connecting two sticky ends (one from the DNA backbone and one from the DNA insert).
Usually, the reaction is catalyzed by the T4 DNA ligase enzyme. It is crucial to respect the vector to insert ratio and therefore the amount of insert must be determined.
Insert amount (ng) = Vector amount x Insert size / Vector size x (Molar ratio)
Molar ratios (Insert/Vector ratio) of 3:1 are used for optimizing cohesive end ligations whilst higher molar ratios are used for blunt end ligations.
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Steps on how to print your input & results:
1. Fill in the calculator/tool with your values and/or your answer choices and press Calculate.
2. Then you can click on the Print button to open a PDF in a separate window with the inputs and results. You can further save the PDF or print it.
Please note that once you have closed the PDF you need to click on the Calculate button before you try opening it again, otherwise the input and/or results may not appear in the pdf.
The Vector Ligation Formula for Optimized Cloning
During the ligation rection, a recombinant plasmid is created from covalently connecting two sticky ends (one from the DNA backbone and one from the DNA insert). Usually, the reaction is catalyzed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together. The vector acts as a carrier of a certain gene or plasmid.
The amount of DNA must be adjusted based on the length of the DNA to get a ratio of (usually) 3 available insert ends for every available vector end.
Insert amount (ng) = Vector amount x Insert size / Vector size x (Molar ratio)
Molar ratios (Insert/Vector ratio) of 3:1 are used for optimizing cohesive end ligations whilst higher molar ratios are used for blunt end ligations.
Ligation is essential in DNA replication in the human body whilst exogen ligation reactions are used to create substances, such as insulin, in gene therapy.
References
Revie D, Smith DW, Yee TW. Kinetic analysis for optimization of DNA ligation reactions. Nucleic Acids Res. 1988 Nov 11;16(21):10301-21.
Cranenburgh RM. An equation for calculating the volumetric ratios required in a ligation reaction. Appl Microbiol Biotechnol. 2004; 65(2):200-2.
Upcroft P, Healey A. Upcroft P, et al. Rapid and efficient method for cloning of blunt-ended DNA fragments. Gene. 1987; 51(1):69-75.
Ng DTW, Sarkar CA. Model-guided ligation strategy for optimal assembly of DNA libraries. Protein Engineering, Design and Selection, Volume 25, Issue 10, October 2012, Pages 669–678.
Article By: Denise Nedea
Published On: August 28, 2020 · 12:00 AM
Last Checked: August 28, 2020
Next Review: August 28, 2025