![]() ![]() ![]() activity A technique for radiolabeling DNA restriction endonu. C) Requires P)-radiolabeling of 5 ends of DNA D). assay to detect the enzymatic activity of the Klenow fragment with the adsorbed DNA to GO. As in the case with the kinase labeling of DNA ( Chapter 39), the DNA fragment is cleaved with a second restriction enzyme, to generate fragments of unequal size that are labeled only in one end.The fragments are subsequently separated on the basis of their different molecular weights and isolated ( chapter 7 and chapter 10). The photoaffinity compound Sazido-dATP was used as a probe for the deoxyribonucleoside triphosphate- binding site of the large fragment of DNA polymerase. A) Single stranded DNA is replicated using a modified DNA polymerase enzyme (e.g., Klenow fragment). The DNA fragment to be labeled is incubated in the presence of one or more deoxyribonucleotide triphosphates (one of which is labeled with 32P in the a-phosphate group) and the Klenow fragment of DNA polymerase. The principle of the method is shown in Fig. The method described here is for generating 3′ end-labeled DNA fragments that are suited for sequencing by the method of Maxam and Gilbert ( Chapter 51), but can in principle also be used when radioactively labeled DNA is required for other purposes (e.g., Southern hybridization). activity varied with each digest, generally accounting for 30. Radiolabeling of DNA fragments is most efficient with DNA fragments that contain recessed 3′ ends ( see Fig. To radioactively label a DNA fragment for use as a probe in blotting. ![]() The fragment still has the polymerase and 3′–5′ exonuclease activity, but lacks the 5′–3′ exonuclease activity of the holoenzyme ( 1). 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 (8). Stop the reaction by heating the mixture for 10 minutes at 75C. Incubate the reaction at room temperature for 10 minutes. The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin. Moreover, there are mounting concerns when it comes to hazardous waste disposal and safety issues associated with radio- activity. unit of Klenow Fragment per microgram of DNA. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |