Cleave DNA Using Restriction Endonulease, A Bacterial Enzyme

In order to manipulate DNA you have to posses the ability to cleave DNA at specific sites by using bacterial enzyme, which is restriction endonulease. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. The name of the enzyme (such as BamHl, EcoRl, AluI, and so on) tells us about the origin of the enzyme but does not give us any information about the specificity of cleavage. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AZul),GAATTC (for EcoRl), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence but cleave the DNA at different points within that sequence.

Agarose Gel Electrophoresis of Nucleic Acids

After amplify DNA template using PCR method, now you can continue your work by using gel electrophoresis in a gel composed of agarose in order to separate DNA fragments based on its molecular weight. The percentage of agarose used depends on the size of fragments to be resolved. In general a 0.8-1% gel may be used for effective separation of DNA fragments of 100-1500 base pairs.

Bacterial DNA Extraction for Pulsed-field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a method used to separate large DNA fragments such as those obtained after digestion with restriction endonucleases that cut infrequently. To avoid possible risks of shearing bacterial DNA during the extraction and digestion steps, bacterial cells are immobilized prior to processing by incorporation in agarose gel. DNA extraction for PFGE is characterized by the need to prolong contact between agarose plugs and the lysis solution that must be distributed throughout the gel. However, the duration of DNA preparation has been shortened since the initial description of the method. The method described in this posting works well with Gram-positive and Gram-negative rods of clinical interest.

Polymerase Chain Reaction (PCR): Basic Principle

Polymerase chain reaction (PCR) is a primer mediated enzymatic amplification of specifically cloned or genomic DNA sequences. PCR process was invented by Kary Mullis and it has been automated for routine use in laboratories worldwide. The main purpose of the PCR process is to amplify template DNA using thermostable DNA polymerase enzyme which catalyzes the buffered reaction in which an excess of an oligonucleotide primer pair and four deoxynucleoside triphosphates (dNTPs) are used to make millions of copies of the target sequence.

The Measurement of Tryptophan Content by Using UV-Spectrometer

The absorption of protein solutions in the UV is the result of tryptophan and tyrosine (and to a very minor, and negligible, extent phenylalanine and cysteine). The absorption maximum will depend on the pH of the solution, and spectrophotometric measurements are usually made in alkaline solutions. Absorption curves for tryptophan and tyrosine show that at the points of intersection, 257 and 294 nm, the extinction values are proportional to the total tryptophan + tyrosine content. Measurements are normally made at 294.4 nm, since this is close to the maximum in the tyrosine curve, and in conjunction with the extinction at 280 nm, the concentrations of each of the two amino acids may be calculated. This is the method of Goodwin and Morton.