What molar ratio should I use for ligation?

Keep total DNA concentration between 1-10 µg/ml. Vector: Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors). Insert: vector molar ratio should be 6:1 to promote multiple inserts. Use NEBioCalculator to calculate molar ratios.

What is ligation ratio?

More the number of insert, higher is the chance of collision with vector. Hence, higher chance of proper ligation. Thus vector to insert ratio is ideally 1:3. Depending on the requirement, it can be changed to 1:5 or even 1:7 to increase chances of getting positive clones.

How do you do ligation on a calculator?

How to use the ligation calculator?

1. Enter your vector mass – in either nanograms (ng) or micrograms (μg).
2. Choose the insert/vector molar ratio. The ideal ratio is 3:1.
3. In this particular situation, your result will be the insert mass needed for the reaction. We recommend that you add at least 50 ng of insert.

Why is ligation done at low temperature?

Here’s why we carrying out DNA Ligation at low temperatures can help. The DNA ligase enzyme has optimal activity at 25°C so the ligation reaction is carried out at a temperature that is a trade-off between the optimal temperatures for bringing the DNA ends together (1°C) and the enzymatic reaction (25°C).

What is a good ligation efficiency?

The ligation efficiency reached maximum in the temperature range of 12–16 °C. The activity of T4 DNA ligase increases with an increase in the temperature up to its optimal temperature (37 °C).

How much DNA is needed for a ligation?

We recommend around 100 ng of total DNA in a standard ligation reaction. Use a ligation calculator to easily quantify how much vector and insert DNA to use.

How do molar ratios work?

Molar ratios state the proportions of reactants and products that are used and formed in a chemical reaction. Molar ratios can be derived from the coefficients of a balanced chemical equation.

How can I increase my ligation efficiency?

Include polyethylene glycol (PEG) PEG is a hydrophobic molecule that takes up space in the reaction, effectively increasing the concentration of the aqueous reaction components e.g. DNA, ATP and ligase. Adding PEG (e.g. PEG 8000) to a final concentration of 5-15% may increase ligation efficiency.

What is the ligation process?

Ligation can be defined as the act of joining, and in biology the term refers to an enzymatic reaction that joins two biomolecules with a covalent bond. Ligation occurs as part of normal cellular processes, such as DNA replication, to repair single and double strand DNA breaks.

How can you increase the efficiency of a ligation?

How long can you store a ligation reaction?

It is always good to store at 4C for 12-36 hours and for long term storage (without repeated freeze and thaw) use -20C.

How to calculate insert size in ligation calculator?

……insert size (in bp): Please enter the molar vector : insert ratio: (normally a vector to insert ratio of 1 to 3 is used of cohesive end ligations. higher molar ratios can be used for blunt end ligations)

How to calculate molar ratio of vector to insert for ligation?

Our BioMath Calculator is an easy way to calculate the molar ratio of vector to insert for ligation. The vector:insert ratio changes, depending on the insert, even if you use the same vector.

How to calculate ligation ratio for insert Promega?

[ (ng of vector × kb size of insert) ÷ kb size of vector] × (molar amount of insert ÷ molar amount of vector) = ng of insert How much 500bp insert DNA needs to be added to 100ng of 3.0kb vector in a ligation reaction for a desired vector:insert ratio of 1:3?

What should the molar ratio be for in fusion?

The molar ratio of insert:vector is set to 2:1, as recommend by our In-Fusion Cloning protocol. 2. The total DNA amount (insert + vector) provided by the calculator is 200 ng, which is optimal for a 10 µl In-Fusion Cloning reaction.