How do you Dephosphorylate DNA?
Procedure:
- Dissolve DNA in 1X CIP Buffer (0.5 µg DNA/10 µL).
- For 5′ overhang DNA add 0.1 units/pmol CIP; for 3′ overhang or blunt end DNA add 1 unit/pmol.
- Incubate 60 minutes at 37 °C.
- Extract with phenol/chloroform2 (Product No. P3803 or P2069) or gel purify the DNA. *
- Recover the DNA by alcohol precipitation.
Why do we use Dephosphorylate plasmid?
Dephosphorylation is a common step in traditional cloning workflows to ensure that the vector does not re-circularize during ligation. If the vector is dephosphorylated, it is essential to ensure that the insert contain a 5′ phosphate to allow ligation to proceed.
Can blunt ends be ligated?
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.
Which are blunt ended DNA molecules?
Blunt ends are also called non-cohesive ends, since there is no unpaired DNA strand fleeting at the end of DNA. The sticky ends, a.k.a. cohesive ends, have unpaired DNA nucleotides on either 5′- or 3′- strand, which are known as overhangs.
Do I need to dephosphorylate my vector?
Dephosphorylation of 5´ Ends For linear vectors with unique 5′ ends, dephosphorylation is not necessary. The dephosphorylation reaction can be performed directly in restriction enzyme buffer so the vector can be cut and dephosphorylated at the same time.
How much linear DNA can be blunted and phosphorylated in one reaction?
Up to 1-5 microgram of the linear DNA can be blunted and phosphorylated in one 20 min reaction. After the thermal inactivation the reaction mixture can be used for blunt-end ligations.
How do you dephosphorylate a protein or DNA?
To dephosphorylate a protein or DNA, an enzyme or hydrolase that cleaves ester bonds is required. For example, phosphatases remove phosphate groups by hydrolyzing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl (−OH) group. 1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase 2.
Why blunting and phosphorylation for DNAs?
With a special blunting and phosphorylation enzyme blend, all kind of DNAs can be treated in one tube in a single procedure to achieve efficient blunting and phosphorylation prior to ligation. The procedure is simple and saves more than 60% of time in comparison to blunting and phosphorylation reactions performed separately.
What is dephosphorylation and how is it used in cloning?
Dephosphorylation is a common step in traditional cloning workflows to ensure that the vector does not re-circularize during ligation. If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, use of a phosphatase, such as Quick CIP, to remove the 5´ phosphate reduces…