What is a Weiss unit?
One Weiss unit is defined as the amount of enzyme required to convert 1 nmol of 32P-labeled inorganic pyrophosphate into Norit adsorbable material in 20 minutes at 37°C, using specified reaction conditions (Weiss, B., et al., (1968) J. Biol. Chem., 243, 4543).
What is the definition of ligase activity1 Weiss unit?
A measure of T4 DNA ligase activity based on ATP‐PPi exchange. One unit is the amount of enzyme required to catalyse the exchange of one nanomole of 32P from 32PPi From: Weiss unit in Oxford Dictionary of Biochemistry and Molecular Biology »
What is the definition of one unit for this enzyme T4 DNA ligase?
Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 ul in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.
What is the definition of one unit of T4 DNA ligase?
Unit Definition: (Cohesive End Ligation Unit): One NEB unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM [300 µg/ml]) in 20 µl of 1X T4 DNA Ligase Reaction Buffer in 30 minutes at 16°C.
How blunt ends are ligated?
Blunt-end ligation Blunt end ligation does not involve base-pairing of the protruding ends, so any blunt end may be ligated to another blunt end. Blunt ends may be generated by restriction enzymes such as SmaI and EcoRV.
What are the optimal reaction conditions recommended by the manufacturer buffer and incubation conditions )?
b) What are the optimal reaction conditions recommended by the manufacturer (buffer and incubation conditions)? The optimal reaction conditions recommended by the manufacturer: incubate at 16C in 1X T4 Ligase Reaction Buffer.
Can T4 ligase ligate blunt ends?
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).
Why sticky ends are better than blunt ends?
Because sticky ends find each other faster due to their attraction for each other, the process of ligation requires less human DNA and less plasmid DNA. The blunt ends of DNA and plasmids are less likely to find each other, and thus ligation of blunt ends requires that more DNA is put into the test tube.
Why do we need blunt ends?
Blunt-end cloning is also one of the easiest and most versatile methods for cloning dsDNA into plasmid vectors. It is easy because the blunt-ended insert requires little to no preparation—avoiding the enzymatic digestion and subsequent purification needed for cohesive-end cloning.
Can you over Ligate for too long?
By the way 1-5 ng of DNA per 50 ul competent cell is sufficient for transformation so I would suggest not to exceed more than 5 ul of ligation reaction. putting too much of reaction may have an inhibitory effect on transformation due to reaction ingredients and salts.
Can blunt ends be ligated?