部分限制酶酶切位点、保护性碱基及酶切效率表doc.pdf

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1、 New England Biolabs Technical Literature-Updated 03/13/2004 部分限制酶酶切位点、保护性碱基及酶切效率表 Cleavage Close to the End of DNA Fragments(oligonucleotides)NOTE:To test the varying requirements restriction endonucleases have for the number of bases flanking their recognition sequences,a series of short,double-st

2、randed oligonucleotides that contain the restriction endonuclease recognition sites(shown in red)were digested.This information may be helpful when choosing the order of addition of two restriction endonucleases for a double digest(a particular concern when cleaving sites close together in a polylin

3、ker),or when selecting enzymes most likely to cleave at the end of a DNA fragment.The experiment was performed as follows:0.1 A260 unit of oligonucleotide was phosphorylated using T4 polynucleotide kinase and g-32P ATP.1 g of 5 32P-labeled oligonucleotide was incubated at 20C with 20 units of restri

4、ction endonuclease in a buffer containing 70 mM Tris-HCl(pH 7.6),10 mM MgCl2,5 mM DTT and NaCl or KCl depending on the salt requirement of each particular restriction endonuclease.Aliquots were taken at 2 hours and 20 hours and analyzed by 20%PAGE(7 M urea).Percent cleavage was determined by visual estimate of autoradiographs.As a control,self-ligated oligonucleotides were cleaved efficiently.Decreased cleavage efficiency for some of the longer palindromic oligonucleotides may be caused by the formation of hairpin loops.