However, in order to generate a 3 blunt end that will be compatible with the HpaI cloning site in the DONAI vector, pEGFP-N1 is digested first with the NotI enzyme followed by filling in the 5’ overhang using Klenow DNA polymerase. 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. Thermo Scientific NotI restriction enzyme recognizes GCGGCCGC sites and cuts best at 37C in O buffer (Isoschizomers: CciNI). The EGFP cDNA can be extracted from pEGFP-N1GFP-N1 by restriction digestion with BamHI and NotI enzymes. 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). The NotI sites are each located a-p proximately 300 bp from theEcoRI cloning site. 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. NotI sites flanking thEc eoRI cloning site of the BAC vector pECSBAC4 (1).
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