What is the restriction site for BglII?

Thermo Scientific BglII restriction enzyme recognizes A^GATCT sites and cuts best at 37°C in buffer O. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.

What is HF enzyme?

Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design. …

What is the recognition sequence for BglII?

BglII’s active site is similar to other endonucleases’, following the sequence Asp-(X)9-Glu-X-Gln. In its active site there sits a divalent metal cation, most likely Mg2 +, that interacts with Asp-84, Val-94, a phosphoryl oxygen, and three water molecules.

How do I cancel BglII?

BglII is not inactivated by incubation at 80°C for 20 min. To prepare the digested DNA for electrophoresis: – stop the digestion reaction by adding 0.5 M EDTA, pH 8.0 (#R1021), to achieve a 20 mM final concentration.

Does EcoRV produce blunt ends?

EcoRV (pronounced eco R five) is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I. In molecular biology, it is a commonly used restriction enzyme. It creates blunt ends.

What do restriction enzymes recognize?

Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions are called recognition sequences, or recognition sites, and are randomly distributed throughout the DNA.

What is the fidelity of polymerase?

The fidelity of a DNA polymerase refers to its ability to accurately replicate a template. … The rate of misincorporation (incorporating the incorrect nucleotide) is known as the polymerase’s error rate.

What is star activity restriction enzymes?

It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed star activity.

Why do I see a DNA smear on an agarose gel after a restriction digest?

The source of nuclease contamination may come from the DNA preparation, the digestion buffer or the water used in the digestion mix. … If the buffer in the gel box appears cloudy and gel runs show abnormal results, rinse the gel box and use a fresh gel before loading your digestion for best results.

What are the differences between Isoschizomers and Neoschizomers enzymes?

The key difference between isoschizomers and neoschizomers is that isoschizomers are restriction enzymes that have the same recognition sequence and cleave the DNA at the same positions, while neoschizomers are restriction enzymes that have the same recognition sequence but cleave DNA at different positions.

Does Hind 3 produce blunt ends?

Option B: Hind 3: It is a type 2 restriction endonuclease which gives sticky ends. It is isolated from Haemophilus influenzae. … Eco RV: It is type 2 endonuclease producing blunt ends in the centre of nucleotide sequence GAT/ATC.

Does Hind 2 produce blunt ends?

Compatible ends Hind II generates fragments with blunt ends and is compatible to any other blunt end.

How efficient is blunt end ligation?

Compared to sticky-end ligations, blunt-end ligations are less efficient, in fact, 10 – 100 times less efficient. This is because, unlike sticky end cloning, there is no hydrogen bonding between the complementary nucleotide overhangs to stabilize the formation of the vector/insert structure.

What are the three types of restriction enzymes?

Today, scientists recognize three categories of restriction enzymes: type I, which recognize specific DNA sequences but make their cut at seemingly random sites that can be as far as 1,000 base pairs away from the recognition site; type II, which recognize and cut directly within the recognition site; and type III, …

What are the two types of restriction enzymes?

Types of Restriction Enzymes

Can restriction enzymes cut RNA?

Restriction enzymes cut DNA* at specific sites based on the sequence of bases along the strand at the cut site. … Restriction enzymes are nucleases – enzymes that cut nucleic acid polymers (i.e. DNA and RNA). There are two types of nuclease: endonuclease and exonuclease. Endonucleases make cuts within a DNA polymer.

What is meant by Replisome?

Definition. The replisome is a large protein complex that carries out DNA replication, starting at the replication origin. It contains several enzymatic activities, such as helicase, primase and DNA polymerase and creates a replication fork to duplicate both the leading and lagging strand.

Which polymerase has the highest fidelity?

DNA polymerase RB69 DNA polymerase like T4 DNA polymerase is a processive and high fidelity enzyme responsible for coordinated replication of both leading and lagging DNA strands.

What is low fidelity DNA polymerase?

These low-fidelity DNA polymerases flout the conventional rules of DNA base pairing. They also may have specialized functions and evolutionary implications that are as yet almost completely unknown. And they may provide a new type of research tool for synthesizing novel forms of DNA to order.

What is star activity example?

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 causes 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 factors contribute to star activity of restriction endonuclease?

These include pH, type of ions present, ionic strength, metal cofactors other than Mg2 +, high enzyme:DNA ratios and the presence of volume excluders (glycerol, ethylene glycol, etc.). In conjunction with this increase in star activity, cleavage rates at the cognate site generally decrease.

What causes DNA smearing during electrophoresis?

1. Improperly prepared gel: If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear. … If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel.

What is the most likely reason that you see a smear of stained DNA after restriction enzyme digestion of the genomic DNA?

With beginning molecular biologists, the most likely reason for the smearing is contamination by some stray nuclease that degraded the DNA into dozens, hundreds, or even thousands of little pieces.

What is a smear on agarose gel?

Gel electrophoresis allows scientists to visualize digested samples and measure the sizes of the fragments. Smearing results from improperly prepared agarose gels, loading an undiluted sample into the wells or using poor quality samples.