Restriction Enzymes NdeI

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 4 matching solutions for this experiment

NdeI (10 U/µL)

Thermo Fisher Scientific

Protocol tips
PCR-amplified fragments were digested with NdeI and XhoI enzymes (Thermo Fisher Scientific Inc.), ligated into the pET28a plasmid vector by using T4 DNA ligase (Thermo Fisher Scientific Inc.) and used to transform E. coli Top10 competent cells to isolate proper clones which were used for protein expression in E. coli BL21 (DE3) cells.
FastDigest NdeI

Thermo Fisher Scientific

Protocol tips
Amplified CdtB gene and vector were digested using thermo fast digest HindIII and NdeI enzymes.
Downstream tips
After digestion, both vector and gene were gel purified and ligation was done using Takara T4 DNA ligase enzyme at 16 °C for 30 min.
Protocol tips
Similarly, the 3′ PpAOS1 genomic fragment was inserted into pTN182-PpAOS1KO5′, which had been digested with SphI (Takara Bio Inc., Shiga, Japan) and NdeI, (Takara Bio Inc., Shiga, Japan) to yield pTN182-PpAOS1KO.
NdeI NEB#R0111

New England BioLabs

Protocol tips
PCR products were digested with NdeI, ApaLI, and AccI (NEB) at 37°C, ethanol-precipitated, and run on denaturing polyacrylamide gels as described in ref. 15.
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