SACI Enzyme | Restriction enzymes bind DNA


The SacI Enzyme

SacI is an essential factor in determining whether a DNA strand is suitable for methylation. It recognizes the cytosine methylation site of a DNA strand and blocks cleavage when two CG overlaps are present. This occurs if C precedes or G follows the recognition sequence. The enzyme will not cleave DNA from a mammalian source or DNA that contains a CG methylase.

SacI is a restriction endonuclease isolated from Streptomyces chromogens ATCC 12767. It was isolated using a procedure that provides a highly electrophoretically homogenous enzyme preparation. The enzyme can be stored at -20 C. For optimal activity, it should be frozen and stored at -20C. Control DNA digestion ensures proper activity, as glycerol can interfere with the reaction.

A host cell culture expressing the sacI restriction endonuclease is required for production. After the cell culture has been transformed, SacI restriction endonuclease can be produced. Once the plasmid has been produced, it can be used for DNA digestion. Once the DNA has been converted, the enzyme is expressed in the host cell. The enzyme is then purified using a specialized enzyme purification process.

Restriction endonuclease for chitosan

The purified enzyme is a particular restriction endonuclease for chitosan. The purified enzyme hydrolyzes chitosan and its derivatives. It does not degrade cellulose or chitin. It is helpful for the biosynthesis of polyethylene glycols. The enzyme is also used in the treatment of septic flora. It is important to note that SacI restriction endonuclease is not a methylase.

The activity of the SacI enzyme has recently been studied in detail using various methods. The first is the co-crystal structure of the enzyme bound to a cognate sequence. The second method is the substitution experiment which helps postulate a mechanism for DNA cleavage. The SacI enzyme is insensitive to adenine methylation but is resistant to the AluI methyltransferase (AGM). The enzyme’s activity is affected by several drugs, including heparin and EDTA.

The SacI enzyme is also available in a High Fidelity form. This enzyme is available from the Promega Company and is qualified as Time-Saver. Its molecular weight is 52,000 D, and its isoelectric point is 6.2. However, this enzyme has two subunits and an isoelectric point of 6.2. The enzyme’s activity can be affected by various factors, including excessive glycerol, magnesium, or manganese. Other potential causes include non-optimal NaCl concentration, extreme pH, and the presence of organic solvents.

BSA is used to stabilize the enzyme

BSA is used to stabilize the enzyme during storage. It also enhances its activity. The enzyme should be stored at -20 C. Repeated freezing and thawing cycles should be avoided to retain optimal activity. When preparing the enzyme for use, it is best to prepare the reaction mix with a particular buffer. This Buffer contains specific salt and pH requirements for optimal activity. In low activity, the enzyme should be stored in a dry, air-tight foam container in the freezer.

StyI is a protein containing a single recognition site, usually cCWWGG. This recognition site is a palindromic sequence; however, substrate sequences are often incompletely palindromic, with one to nine unspecified nucleotides. StyI recognizes the substrate sequence in the presence of a cleavage-friendly sequence. This recognition site may influence the enzyme’s kinetics.

Thermo Scientific SacI enzyme recognizes the GAG CTC site in DNA and cuts best at 37degC in its Buffer. The enzyme’s Reaction Conditions for Restriction Enzymes list the conditions required for double digestion and heat inactivation. FastDigest enzymes are available for the rapid digestion of DNA. Unless the enzyme is specifically designed for fast DNA digestion, it can be substituted with commercially available DNA.

Restriction enzymes bind DNA

Restriction enzymes bind DNA both specifically and nonspecifically. EcoRI and EcoRV, two restriction enzymes, diffuse along with linear DNA at 1.7 x 106bp s-1, respectively. Water molecules fill the space between the enzyme and DNA during this process, which is excluded once the cognate sequence is found. During this process, many contacts develop between the enzyme and DNA bases and the phosphodiester backbone.

The SacI restriction endonuclease gene sequence is illustrated in Fig. 2 and corresponds to the SacI methylase gene. SacI has an open reading frame of 804 bp and has 27 amino acid identities with the scale protein. PCR determined the DNA sequences, and short PCR products were cloned into pUC19.

For optimal activity, the enzyme should be stored at -20C. Multiple freeze-thaw cycles should be avoided to maintain optimal activity. Using control DNA digestion is essential to ensure adequate activity, as the enzyme contains a high glycerol content. For best results, aliquot samples as needed and store them on ice until use. The enzyme should be kept in a foam storage container in the freezer during long-term storage.

SacI-HF is sensitive to overlapping

SacI-HF is sensitive to overlapping cytosine methylation. It blocks cleavage when there is a double CG overlap and when C precedes the recognition sequence. Nonetheless, the enzyme remains active if the DNA molecule comes from a mammalian source or a host containing CG methylase. For the most effective use, a sacI-HF enzyme synthesis reaction requires three times the amount of heparin.

SDI is an effective restriction enzyme, as it recognizes GAG CTC sites. It cuts DNA best at 37°C in its Buffer. The Reaction Conditions of Restriction Enzymes list conditions for optimal activity of the enzyme, including temperature and double digestion. It is also available as a FastDigest enzyme for rapid DNA digestion. Use the SacI restriction enzyme before preparing your DNA reaction to prevent degradation.

A comparative study of Saci enzyme and SspI reveals that SspI has a more favorable substrate specificity. Using 1.2 mL of PBX DNA as a substrate, SacI cleaves it with high fidelity. The enzyme can also recut DNA fragments and ligate them. However, its catalytic activity is unrelated to its affinity.

Although there are over 3,000 restriction enzymes, they all have 200 different target sequences. As such, the difference between the cleavage rates of SspI and EcoRI enzymes is high under optimal conditions. EcoRI cleaves DNA at the related site (5′-GAATTC-3′) 105 times faster than the following best site, and EcoRV cleaves it 106 times faster.

The GAG CTC site and cuts DNA best at 37 degC

The SacI restriction enzyme recognizes the GAG CTC site and cuts DNA best at 37 degC in its unique Buffer. The SacI enzyme’s Reaction Conditions list the conditions for double digestion, heat inactivation, and other factors. The SacI enzyme is also available as a FastDigest enzyme for rapid DNA digestion. For more information, see the SacI enzyme’s Product Sheet. This enzyme can be used for DNA digestion in various applications, including the synthesis of cDNA.

The SacI methylase gene is cloned after the SacI endonuclease gene is cloned. Usually, the SacI methylase gene is present next to the SacI endonuclease gene. When the cloned DNA is resistant to SacI digestion, it is analyzed to identify the presence of a SacI methylase gene. After digestion, the scale gene and adjacent DNA are sequenced to identify the SacI methylase gene.

The High Fidelity SacI enzyme is a modified SacI with 100% activity in the rCutSmart Buffer. This enzyme has dramatically reduced star activity and is Time-Saver-qualified. This enzyme has the same fidelity as the native SacI enzyme but has improved flexibility in experimental design. Its high-fidelity properties make it a valuable tool for biotechnological research. It has become a popular tool for biotechnology researchers.


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