If DNA pol I was used in this example, it could degrade (5'-3' exnuclease) some of these strands (ones that did not have a primer attached).
SHELF LIFE OF KLENOW FRAGMENT FREE
The only reasoning I could come up with was, with the use of denaturing dsDNA, there would be free floating ssDNA. Note: Klenow Fragment, Exonuclease Minus, will leave a single-base 3-overhang for a significant proportion of the DNA fragments during the fill-in reaction (5). Stop the reaction by heating the mixture for 10 minutes at 75C. Incubate the reaction at room tempera-ture for 10 minutes. However, when it comes to primer extension radio labeling (using a single strand as the template - cDNA, or maybe even denaturing of dsDNA?), the klenow fragment is used.why? 1 unit of Klenow Fragment per microgram of DNA. It seems when it comes to nick translation radiolabeling, DNA pol I is used understandable since the 5'-3' exonuclease is needed to degrade the non-template strand, allowing for the radiolabeling. My question revolves around radioactive labeling and its applications. Degrade 3' over hangs (again it seems DNA pol I could do this, or would the 5'-3' exonuclease begin to degrade the opposite strand?) Fill in 5' over hangs (Can't DNA pol I do this?) Many sources say klenow fragments are able to synthesize dsDNA from cDNA (make dsDNA from a single strand) but can't DNA pol I do this? (of course both needing primers).Īdditionally, it is pointed out that Klenow fragments can be used to form blunt ends: My question though stems from many of the sources I've been scouring. Some more information regarding my question:įrom my understanding, the only difference that a klenow fragment has over DNA polymerase I, is its removal of 5'-3' exonuclease activity (additionally, removal of 3'-5' exonuclease activity if specified).
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