How efficient is blunt end ligation?

Compared to sticky-end ligations, blunt-end ligations are less efficient, in fact, 10 – 100 times less efficient. This is because, unlike sticky end cloning, there is no hydrogen bonding between the complementary nucleotide overhangs to stabilize the formation of the vector/insert structure.

Which ends have more efficient ligation?

The chemical reaction involved in ligation of two molecules is same as repairing discontinuities. Explanation: In the case of blunt ends the ligation is less efficient because ligase cannot catch hold of the molecules to be ligated, and has to wait for chance associations to bring the ends together.

How can I improve my ligation?

Our top 5 DNA ligation tips should improve the efficiency of your ligations, and will hopefully increase your cloning success rate!

1. Aliquot the ligase buffer.
2. Heat the DNA just before ligation.
3. Check the pH.
4. Include polyethylene glycol (PEG)
5. Add a restriction enzyme just before transformation.

What ligation ratio should I use?

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.

Is it possible to perform a blunt end ligation?

For more information about New England Biolabs visit neb. Blunt-end cloning involves the ligation of DNA fragments – usually between a plasmid vector and an insert – whose terminal ends are not “sticky”. Performing these ligations is notoriously difficult, particularly with large DNA fragments. But it is possible.

Why do you need more ligase in blunt end cloning?

This is because, unlike sticky end cloning, there is no hydrogen bonding between the complementary nucleotide overhangs to stabilize the formation of the vector/insert structure. For this reason, a blunt-end ligation requires more ligase, and more of your linearized vector and insert.

Which is the best ligase for blunts and overhangs?

For blunt and single-base overhangs, Blunt/TA Ligase Master Mix is recommended. For ligations that are compatible with electroporation, ElectroLigase (NEB #M0369) is recommended. Standard T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes. Do not heat inactivate the Quick Ligation Kit or ligase master mixes.

Can You ligate one blunt end and one sticky end?

With one blunt end and one sticky end, is it necessary to dephosphorylate the vector and phosphorylate the insert so that the blunt ends ligate? In my experience, ligations with one sticky and one blunt end work very efficiently, not that different from two sticky ends.