Can T4 ligase ligate blunt ends?

Can T4 ligase ligate blunt ends?

Catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids (1).

How do you use T4 DNA Ligase?

How to set up a ligation reaction using T4 DNA ligase

1. Into a reaction tube, combine the following: vector, insert DNA, T4 Ligase Buffer, T4 DNA Ligase, and nuclease-free water.
2. Gently mix the reaction and centrifuge briefly.
3. Incubate the reactions.
4. Heat-inactivate the ligation reactions.

Can DNA ligase connect blunt end fragments?

Blunt-ended fragments can be joined to each other by DNA ligase. However, blunt-ended fragments are harder to ligate together (the ligation reaction is less efficient and more likely to fail) because there are no single-stranded overhangs to hold the DNA molecules in position.

How do you connect blunt ends?

You can create blunt ends by filling in single stranded overhangs remaining after physically shearing (see Fig. 2) or cutting with restriction endonucleases that generate sticky ends. The single-stranded overhangs can be repaired using a mixture of DNA polymerases such as T4 polymerase and the Klenow fragment.

How do you use T4 DNA ligase?

For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes.

How do I ligate blunt and cohesive ends?

For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB’s Quick Ligation Kit (#M2200S) [30 reactions] or (#M2200L) [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

What is the ligation protocol with T4 DNA ligase (m0202)?

Ligation Protocol with T4 DNA Ligase (M0202) Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last.

What is the molar ratio for ligation of T4 DNA?

(T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios. * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. Gently mix the reaction by pipetting up and down and microfuge briefly.