What is the recognition sequence of Hind 3?
Under the standard reaction conditions, the restriction endonuclease HindIII cleaves double-stranded DNA, within the recognition sequence–A/AGCTT–at the position indicated by the arrow. In the presence of dimethyl sulfoxide the substrate specificity of this enzyme is reduced and cleavages occur at additional sites.
Does HindIII have star activity?
Star activity is the relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimal for the enzyme. Star activity can happen because of presence of Mg2+, as is seen in HindIII, for example. …
What kind of enzyme is HindIII?
HindIII (pronounced “Hin D Three”) is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis.
What is the recognition sequence of Hind 2?
Hind II recognizes the sequence GTPy/PuAC and generates fragments with blunt ends (1). Compatible ends Hind II generates fragments with blunt ends and is compatible to any other blunt end. Isoschizomers Hind II is an isoschizomer to Hinc II. Hind II is inhibited by 6-methyladenine as indicated (*).
What is the difference between EcoRI and HindIII?
The major difference between the two is that EcoRI leaves staggered ends, whereas SmaI leaves blunt ends. Staggered ends are much easier to manipulate during cloning because of the base-pairing capacity inherent in them. (Solution from Diane K. Lavett.)
What could cause star activity?
Generally, star activity (i.e., the enzyme cuts a sequence that is an imperfect match to its known target) is caused by nonoptimal reaction conditions.(1),(2) These include: High concentration of glycerol (>5% v/v). High ratio of enzyme units to micrograms of DNA.
What is Type 2 restriction enzyme?
Type II restriction enzymes are the familiar ones used for everyday molecular biology applications such as gene cloning and DNA fragmentation and analysis. These enzymes cleave DNA at fixed positions with respect to their recognition sequence, creating reproducible fragments and distinct gel electrophoresis patterns.
What is the length of recognition sequence of Hind II?
six base pairs
Hence, the restriction endonuclease Hind II always cuts the DNA molecules at a particular point by recognizing a specific sequence of six base pairs.
What is H in Bam h1?
BamHI (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site. This allows the DNA to maintain its normal B-DNA conformation without distorting to facilitate enzyme binding.
How long does it take to digest FastDigest HindIII rev11?
• Incubation at 37°C. • 1 µL of FastDigest HindIII is formulated to digest up to: – 1 µg of lambda DNA in 5 min. – 1 µg of plasmid DNA in 15 min. – 0.2 µg of PCR product in 20 min. – 1 µg of genomic DNA in 10 min, or 5 µg of genomic DNA in 30 min. Thermal Inactivation: Incubation at 80°C for 10 min.
When to use Thermo Scientific FastDigest HindIII buffer?
Thermo Scientific FastDigest HindIII restriction enzyme recognizes A^AGCTT site and cuts best at 37°C in 5–15 minutes using universal FastDigest Buffer. Thermo Scientific FastDigest HindIII is one of an advanced line of fast restriction enzymes that are all 100% active in the universal FastDigest and FastDigest Green reaction buffers.
How to use Labaid FastDigest restriction enzymes for research?
1. Mix gently and spin down. 2. Incubate at 37°C in a heat block or water bath for 5 min.† 3. (Optional) Inactivate the enzyme. 4. If FastDigest Green Buffer was used, load a portion of the reaction mixture directly onto a gel. LABAID FastDigest restriction enzymes For Research Use Only.
When to use FastDigest buffer with no star activity?
Short protocol times with no star activity FastDigest enzymes in combination with FastDigest (Green) Buffer are designed to eliminate star activity: Short incubation time (5–15 min), no need for prolonged digestions. Universal buffer with optimal pH and salt concentration for all enzymes.
