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Removal of 3´ overhangs to form blunt ends.Synthesis of probes by random primers labeling method (2).

This is a cost effective alternative to the Gibson Assembly Method. Applications: Fill-in of 5´ overhangs (1). Effective, ligation free, seamless cloning for overlapping double stranded DNA, using the Klenow fragment. Fill-in of 5´ overhangs to form blunt ends 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.One unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material in 30 minutes at 37C. 1st Ligation: Use the biotinylated dATP and the Klenow fragment to biotinylate and insert an A-tail in the. Klenow Fragment (Large Fragment E.coli DNA Polymerase I) 200 u. coli strain carrying the Klenow Fragment gene. DNA sequencing by the Sanger dideoxy method Principle The protein is produced by a recombinant E.The other small fragment has 5'→ 3' exonuclease activity, but lacks 5'→ 3' polymerase activity and 3'→ 5' exonuclease activity of klenow fragment. It has 3'→ 5' exonuclease activity for proof reading activity This klenow fragment lacks 5'→ 3' exonuclease activity. On the left side, upon treatment with protease, two fragments are formed, a large fragment called the klenow fragments and a small fragment with 5'→ 3' exonuclease activity. On the right side is the DNA Polymerase I before treating with protease. Refer this figure for clear understanding.