#Shelf life of klenow fragment free#

The reaction proceeds are depicted below: To use Klenow to synthesize a complementary strand of DNA, one simply mixes single-stranded template (usually denatured double-standed DNA), primers and the enzyme in the presence of an appropriate buffer (most restriction enzyme buffers work well). The oligonucleotides must be complemenary to some section of template DNA. The primers used for most in vitro polymerization reactions are single-stranded DNAs, typically 6 to 20 bases in length, called oligonucleotides.

#Shelf life of klenow fragment free#

Performing that task in the lab is integral to such processes as synthesizing the second strand DNA in cDNA cloning and generating radioactive probes for hybridization reactions.ĭNA polymerases require a primer to provide a free 3' hydroxyl group for initiation of synthesis. Synthesis of double-stranded DNA from single-stranded templates: The function of DNA polymerases is to synthesize complementary strands during DNA replication. Klenow fragment is useful for several tasks: The enzyme you purchase is almost certainly produced in this manner. In addition to generating Klenow fragment by proteolysis, it can be expressed in bacteria from a truncated form of the DNA polymerase I gene. The large or Klenow fragment of DNA polymerase I has DNA polymerase and 3' -> 5' exonuclease activities, and is widely used in molecular biology. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5' -> 3' exonuclease activity, and a large piece called Klenow fragment.

However, this pesky enzymatic activity can readily be removed from the holoenzyme. coli's DNA polymerase I makes it unsuitable for many applications.