Restriction Enzymes BssHII / PteI

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 inclue: 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 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, 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 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

FastDigest PteI

Thermo Fisher Scientific

Protocol tips
The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped.
Protocol tips
The restriction enzymes used were AluI (TaKaRa) for Glu178Glu (reference SNP no. rs45483293) BssHII (TaKaRa) for Ala196Ala (rs5480) BsmAI (New England BioLabs, Beverly, MA, USA) for Ser180Phe (found in a Japanese patient with apparent mineralocorticoid excess) and HhaI (TaKaRa) for Arg208His (rs28934592) within exon 3 and HhaI for Arg279Cys (rs28934594) within exon 5. Among these four polymorphisms, the HapMap frequency for JPT is available only with Ala196Ala and is reported as 0.
BssHII NEB#R0199

New England BioLabs

Protocol tips
The PCR products were taken through a phenol/chloroform extraction and then ethanol precipitated prior to being digested for 2 h with the restriction enzyme BssHII (NEB). Loading dye (super orange G) was added to the restriction digest and the sample was loaded into a 4% agarose gel.
PauI (BssHII) (10 U/µL)

Thermo Fisher Scientific

Protocol tips
These plasmids were digested with ClaI and PauI (Thermo Fisher Scientific) and the Pbp1 polyglutamine sequence was gel purified and ligated into the full length Pbp1+ promoter plasmid. All plasmids had sequence fidelity confirmed by Sanger sequencing.
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