Cleavage Clo to the End of DNA Fragments (linearized vector) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Linearized vectors were incubated with the indicated enzymes (10 units/µg) for 60 minutes at the recommended incubation temperature and NEBuffer for each enzyme. Following ligation and transformation, cleavage efficiencies were determined by dividing the number of transformants from the digestion reaction by the number obtained from religation of the linearized DNA (typically 100-500 colonies) and subtracting from 100%. "Ba Pairs from End" refers to the number of double-stranded ba pairs between the recognition site and the terminus of the fragment; this number does not include the single-stranded overhang from the initial cut. Since it has not been demonstrated whether the single-stranded nucleotides contribute to cleavage efficiency, 4 bas should be added to the indicated numbers when designing PCR primers. Average efficiencies were rounded to the nearest whole number; experimental variation was typically within 10%. The numbers in parenthes refer to the number of independent trials for each enzyme tested (from Moreira, R. and Noren, C. (1995), Biotechniques, 19, 56-59). Note: As a general rule, enzymes not listed below require 6 bas pairs on either side of their recognition site to cleave efficiently. | A | B | E | H | K | M | N | P | S | X |
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New England Biolabs Technical Literature - Updated 03/05/2004 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cleavage Clo to the End of DNA Fragments (oligonucleotides) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
To test the varying requirements restriction endonucleas have for the number of bas flanking their recognition quences, a ries of short, double-stranded oligonucleotides that contain the restriction endonuclea recognition sites (shown in red) were digested. This information may be helpful when choosing the order of addition of two restriction endonucleas for a double digest (a particular concern when cleaving sites clo together in a polylinker), or when lecting 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 kina and γ-[32P] ATP. 1 µg of 5´ [32P]-labeled oligonucleotide was incubated at 20°C with 20 units of restriction endonuclea 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 endonuclea. 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, lf-ligated oligonucleotides were cleaved efficiently. Decread cleavage efficiency for some of the longer palindromic oligonucleotides may be caud by the formation of hairpin loops. | A | B | C | E | H | K | M | N | P | S | X |
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