Cleavage Close to the End of DNA Fragments (oligonucleotides) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
To test the varying requirements restriction endonucleases have for the number of bases flanking their recognition sequences, a series of short, double-stranded 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 polylinker), 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 γ-[32P] ATP. 1 µg of 5´ [32P]-labeled oligonucleotide was incubated at 20°C with 20 units of restriction 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. | A | B | C | E | H | K | M | N | P | S | X |
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Cleavage Close 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%. "Base Pairs from End" refers to the number of double-stranded base 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 these single-stranded nucleotides contribute to cleavage efficiency, 4 bases 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 parentheses 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 bases 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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