What is Klenow fragment of DNA polymerase?

DNA Polymerase I, Large (Klenow) Fragment is a DNA polymerase enzyme that lacks the 5′ to 3′ exonuclease activity of intact DNA Polymerase I, but does exhibit the 5′ to 3′ DNA polymerase and 3′ to 5′ exonuclease activities. Applications: Fill-in of 5´ overhangs (1).

How does the Klenow fragment differ from the intact E coli DNA polymerase I?

The key difference between Klenow fragment and DNA polymerase 1 is that Klenow fragment is a large portion of DNA polymerase 1 which lacks 5′ to 3′ exonuclease activity while DNA polymerase is an enzyme of E. coli which has all three domains including 5′ to 3′ exonuclease activity.

What is the function of the Klenow fragment?

The Klenow fragment is extremely useful for research-based tasks such as: Synthesis of double-stranded DNA from single-stranded templates. Filling in receded 3′ ends of DNA fragments to make 5′ overhang blunt. Digesting away protruding 3′ overhangs.

How does T4 DNA polymerase work?

T4 DNA Polymerase catalyzes the 5’→3′ incorporation of nucleotides into double-stranded DNA using single-stranded and primed DNA as a template. It possesses strong 3’→5′ exonuclease (proofreading) activity but does not exhibit 5’→3′ exonuclease activity. These DNA products are often used in blunt cloning.

How is DNA polymerase I, large ( Klenow ) fragment derived?

DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ —> 5’ exonuclease activity Need a custom/large volume order? Contact Us Bulk packaging may also be available and requested for large recurring orders.

Can a Klenow fragment be used for DNA ligation?

Klenow Fragment, exo- is not recommended for DNA blunting reactions prior to DNA ligation since it frequently adds one or more extra nucleotides to the 3′-terminus of blunt-end DNA substrates in a non-template directed fashion. For Research Use Only.

How does Klenow maintain the fidelity of the holoenzyme?

Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini. An E. coli strain that contains the E. coli polA gene that has had its 5’→3′ exonuclease domain removed. One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.

What kind of buffer to use for Klenow?

Klenow can be used to “fill in” 5′ overhangs of double-stranded DNA fragments using common restriction endonuclease buffers. We recommend REact 2 buffer (1X concentration is 50 mM Tris-HCl pH 8, 50 mM NaCl, 10 mM MgCl2) , but other buffers will also work.