酶切常见问题（Frequent problems with enzyme digestion）.docVIP

酶切常见问题（Frequent problems with enzyme digestion） Frequent problems with enzyme digestion Question: DNA was not cut by an enzyme at all Reason： 1) endonuclease inactivation 2) DNA impure, containing SDS, phenol, EDTA and other enzyme inhibitory factors 3) condition discomfort (reagent, temperature) 4) the base sites on the DNA cleavage site are methylated 5) there is no methylation on the DNA cleavage site (e.g., Dpn, I) 6) there are other modifications on the DNA locus 7) DNA does not exist in this enzyme recognition sequence Countermeasure: 1) standard substrate assay enzyme activity 2) DNA column was purified and ethanol precipitated DNA 3) check the reaction system for the best 4) changing the enzyme hydrolysis of DNA methylation insensitive to dam-, and converting the plasmid DNA to the bacterial strain of dcm- and genotype 5) digestion of DNA with different cleavage non methylated sites (such as San3A I instead of Dpn I) was used to re transfer the plasmid to dcm+ dam+ strain 6) mix the DNA substrate with lambda DAN for cutting verification 7) for other enzymes cut DNA or excessive enzyme digestion to verify Problem: incomplete DNA cutting Reason： 1) decrease of endonuclease activity 2) endonuclease dilution is incorrect 3) DNA impurity, poor reaction condition 4) the base on the DNA site of the endonuclease recognition is methylated or otherwise modified 5) some DNA solutions stick to the tube wall 6) the enzyme solution viscosity is large, sampling is not allowed 7) DNA end annealing after enzyme digestion 8) the enzyme is denatured because of the reaction solution, temperature and strong oscillation 9) excessive dilution reduces enzyme activity 10) the reaction condition is not suitable 11) the nucleic acid sequences that affect the enzymatic efficiency were inserted on both sides of the recognition site Countermeasure: 1) 5-10 times the amount of excess digestion 2) dilution enzyme with enzyme storage solution or reaction buffer 3) ibid 4) ibid 5) a few seco