Restriction Enzymes NheI

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. 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 most common challenges with restriction digest include- 1. inactivation of the enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). 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. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. 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.

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Found 5 matching solutions for this experiment

NheI NEB#R0131

New England BioLabs

Protocol tips
Following isolation from E. coli strain Top10 using a Plasmid Midi Kit (Qiagen, Valencia, CA) according to manufacturer protocols, plasmid pXG-10 [54] was digested with AatII and NheI restriction endonucleases (New England BioLabs, Ipswich, MA) to remove the PLtetO-1 promoter and lacZ fragment.
The amplified product was purified using a QIAQuick gel extraction kit (Qiagen) and then digested with AatII and NheI endonucleases and cloned into the digested pXG-10 plasmid backbone to create pshuA-gfp.
FastDigest NheI

Thermo Fisher Scientific

Protocol tips
The amplified PCR product was digested with SacI at the 5′ and with NheI at the 3′ end followed by ligated into SacI+NheI (FastDigest®, Fermentas, USA) digested pSY-B-MCS plasmid to yield a
construct pSY-B-GFP possessing GFP gene between the MP and CP region (Fig. 2A).
Downstream tips
Recombinant clones were confirmed through colony PCR with GFP gene-specific primers,
restriction digestion and nucleotide sequencing.
NheI (10 U/µL)

Thermo Fisher Scientific

Protocol tips
For the bacterial expression of glutathione S-transferase (GST) fusion products, ORF fragments were excised from the phagemid DNA with the restriction endonucleases PteI and NheI (Thermo Fischer Scientific), subcloned into a custom-designed pGEX-Flag expression vector (36), and grown in a minifermenter as previously described in Deantonio et al. (50).
NheI-HF®

New England BioLabs

Protocol tips
After the amplification, the DNA fragment and the pDR111 vector were digested with NheI-HF and SalI-HF restriction enzymes (NewEngland BioLabs), followed by ligation.
Protocol tips
The plasmid was digested by NheI (TaKaRa) and HindIII to confirm the correct construction of pcDNA3.1-GLUT1 (Figure 1).
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