All Fermentas DNA/RNA modifying enzymes and other proteins are produced under the ISO 9001:2000 quality management system and are subjected to extensive quality control. As a result of these stringent conditions of product analysis, the entire Fermentas product line meets the Fermentas PureExtreme® standard - it is your guarantee of the industry’s highest quality and performance. Our products are monitored for the accuracy of their activity units, the absence of contaminant activities (nucleases, phosphatases and proteases) and for their performance in specific functional tests. A warranty is assigned and an expiry date is listed both on the product label and in the Certificate of Analysis supplied with each product. Product lots are monitored regularly to ensure that they continue to meet the quality control specifications right up to their expiry date.
The stringent quality control procedures at Fermentas consistently guarantee the highest quality of DNA/RNA modifying enzymes and other proteins.
Activity AssaysActivity unit definitions for Fermentas DNA/RNA modifying enzymes are those commonly used in molecular biology. Activity unit definitions and descriptions of reaction conditions are provided in the catalog entry for each product. Reaction conditions may differ for specific research applications.
Substrates for Contaminant Activities AssaysNucleolytic activities are assayed with the following substrates: covalently closed circular DNA (Endodeoxyribonuclease Assay), lambda phage or plasmid DNA fragments (Exodeoxyribonuclease Assay, Blue/White Cloning Assay), [3H]-labeled DNA (Exodeoxyribonuclease Assay), [3H]-labeled RNA (Ribonuclease Assay). [5'-32P]-labeled synthetic single- and double-stranded oligonucleotides are used for the identification of endo-, exodeoxyribonucleases and phosphatases (Labeled Oligonucleotide (LO) Test). The use of labeled oligonucleotides enables detection of even trace amounts of deoxyribonucleases and phosphatases that cannot be detected by the conventional methods. Substrate selection for the evaluation of contaminants depends on specific properties of a particular enzyme.
Double-stranded Endodeoxyribonuclease Assay (dsENDO)The assay is performed in a 50 µl aliquot of reaction buffer supplemented with an enzyme, and with 1 µg of covalently closed circular (supercoiled) DNA (either pUC19 DNA or PhiX174 RF1 DNA). After incubation under the appropriate conditions, the DNA is analyzed on a 1% agarose gel. The product passes this quality control test if neither nicked DNA nor linear DNA is detected.
Single-stranded Endodeoxyribonuclease Assay (ssENDO)The assay is performed in a 50 µl aliquot of reaction buffer supplemented with an enzyme, and with 1 µg of covalently closed circular single-stranded DNA of M13mp19. After incubation under appropriate conditions, the DNA is analyzed on a 1% agarose gel. The product passes this quality control test if no decrease in the amount of closed circular DNA is observed.
Exodeoxyribonuclease Assay I (EXO I)The assay is performed in a 50 µl aliquot of reaction buffer supplemented with an enzyme, and with 1 µg of sonicated [3H]-labeled DNA from E.coli. After incubation under the appropriate conditions, the DNA is precipitated with trichloroacetic acid and the radioactivity of the supernatant is determined. Exodeoxyribonuclease activity is expressed as the percent of total DNA radioactivity released into the acid soluble fraction. The product passes this quality control test if less than 0.5% of the DNA is degraded.
Exodeoxyribonuclease Assay II (EXO II)The assay is performed in a 50 µl aliquot of reaction buffer supplemented with an enzyme, and with 1 µg of either the lambda DNA or plasmid DNA fragments. After incubation under appropriate conditions, the DNA is analyzed on an agarose gel. The product passes this quality control test if DNA fragments are not degraded.
The LO test is a unique assay that allows identification of trace contaminants (endodeoxyribonucleases, exodeoxyribonucleases and phosphatases) in enzyme preparations that are missed by other assays. The assay is performed in reaction buffer containing a particular enzyme or other protein of interest and 5'-[32P]-labeled synthetic oligonucleotides (single-stranded and double-stranded). After incubation under the appropriate conditions, reaction products are separated on a polyacrylamide gel and then analyzed by phosphoimaging. The product passes this quality control test if there is no degradation of both the single-stranded oligonucleotide and double-stranded oligonucleotide.
Blue/White Cloning Assay (B/W)This assay is used to ensure that Fermentas enzyme preparations are free from contaminating activities that may affect the integrity of DNA ends and, therefore, the efficiency of cloning experiments. Linearized DNA molecules containing 5'-overhang, 3'-overhang or blunt ends are used as the assay substrates. To generate such molecules, the pUC57 DNA is cut with either HindIII or PstI, or SmaI restriction enzymes, respectively, within the lacZ reporter gene. The linearized plasmid DNA is then incubated with an enzyme under the appropriate conditions. Then the plasmid DNA is self-ligated to restore its circular structure. The ligated plasmid DNA is used to transform the E.coli XL1-Blue competent cells, which are subsequently plated onto X-Gal/IPTG/Amp agar. Cells which received a plasmid copy with the intact lacZ gene produce blue colonies. If the termini of the linearized pUC57 DNA are altered by contaminating exonucleases and/or polymerases, the lacZ reading frame is interrupted resulting in the appearance of white colonies. The product passes this quality control test if the number of white colonies does not exceed 3%. Details of the assay are provided in the Certificate of Analysis supplied with each product.
Ribonuclease Assay (RNase)The assay is performed in a 50 µl reaction mixture containing enzyme or protein, buffer and 1 µg of [3H]-labeled RNA. After incubation under appropriate conditions, the RNA is precipitated with trichloroacetic acid and the radioactivity of the supernatant is evaluated. Ribonuclease activity is expressed as a percent of total RNA radioactivity released into the acid soluble fraction. % of RNA degradation are given in the Certificate of Analysis of the product.
Protease AssayThe assay is performed in a 200 µl aliquot of 10 mM Tris-HCl buffer (pH 7.5) supplemented with an enzyme, and with 200 µg of azocasein. After incubation under the appropriate conditions, the reaction is terminated with trichloroacetic acid and the absorbance of the supernatant is measured at 400 nm. The product passes this quality control test if azocasein is not degraded.
Functional AssaysAssays performed for a particular modifying enzyme or other protein are indicated both in the catalog entry and in the Certificate of Analysis supplied with each product.
DNA/RNA modifying enzymes should be stored at -20°C.
During shipment on dry ice, enzymes may freeze. This does not affect their quality, because all Fermentas enzymes are 100% active after at least three freeze-thaw cycles.
For 24-48 hour delivery, enzymes may be shipped on blue ice, because their quality is not affected by short exposure to +4°C.
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Updated gegužės 10, 2007 10:05
