102000016397 methyltransferase family Human genes 0.000 claims abstract description 5.239000012505 Superdex™ Substances 0.000 claims abstract description 5.230000014509 gene expression Effects 0.000 claims abstract description 22.108090000623 proteins and genes Proteins 0.000 claims abstract description 26.102000004169 proteins and genes Human genes 0.000 claims abstract description 26.238000004519 manufacturing process Methods 0.000 title claims abstract description 11.241000588724 Escherichia coli Species 0.000 title claims abstract description 16.108091007521 restriction endonucleases Proteins 0.000 title claims abstract description 39.Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.) Filing date Publication date Application filed by Universal Biological Systems (anhui) Co Ltd filed Critical Universal Biological Systems (anhui) Co Ltd Priority to CN201710301236.2A priority Critical patent/CN106967743A/en Publication of CN106967743A publication Critical patent/CN106967743A/en Status Pending legal-status Critical Current Links Original Assignee Universal Biological Systems (anhui) Co Ltd Priority date (The priority date is an assumption and is not a legal conclusion. Universal Biological Systems (anhui) Co Ltd Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.) Inventor 李森 雍德祥 邹刚刚 王方平 刘宗文 Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.) Pending Application number CN201710301236.2A Other languages Chinese ( zh)
Noti enzyme pdf#
Google Patents A kind of production method of restructuring NotI restriction enzymes in Escherichia coliĭownload PDF Info Publication number CN106967743A CN106967743A CN201710301236.2A CN201710301236A CN106967743A CN 106967743 A CN106967743 A CN 106967743A CN 201710301236 A CN201710301236 A CN 201710301236A CN 106967743 A CN106967743 A CN 106967743A Authority CN China Prior art keywords noti restriction enzymes methylase restructuring expression Prior art date Legal status (The legal status is an assumption and is not a legal conclusion. Google Patents CN106967743A - A kind of production method of restructuring NotI restriction enzymes in Escherichia coli To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.CN106967743A - A kind of production method of restructuring NotI restriction enzymes in Escherichia coli Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, the optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with the activity of the other. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. The most common challenges with restriction digest include- 1. The four most common types of restriction enzymes include: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). Restriction Enzymes NotI A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence.