Fermentas - the Leader in
Restriction Enzyme Quality,
flyer in pdf, 183 KBFermentas restriction enzymes are produced under the ISO 9001:2000 quality management system, which combined with our own extensive quality control tests, guarantees consistent PureExtreme® Quality - the highest quality and performance - for the entire Fermentas product line. Fermentas restriction enzymes pass all standard quality control assays, as well as our unique Labeled Oligonucleotide (LO) test which is the most sensitive test for the detection of trace activities of endodeoxyribonucleases, exodeoxyribonucleases and phosphatases.
We monitor all enzyme lots to ensure they meet these stringent quality control specifications right up to their expiry date.
The PureExtreme® Quality of restriction enzymes ensures that the integrity of your DNA is not compromised making them the enzymes of choice for even the most demanding applications.
One unit of restriction enzyme is the amount of enzyme required to hydrolyze 1 µg of substrate DNA in 60 min in 50 µl of reaction mixture under recommended conditions. To determine restriction enzyme activity, concentrated enzymes are first diluted to approximately 500-1000 units/ml with enzyme dilution buffer (20 mM potassium phosphate (pH 7.4), 200 mM KCl, 1 mM EDTA, 7 mM 2-mercaptoethanol, 10% glycerol and 0.2 mg/ml BSA).
In general, enzymes are assayed with lambda phage DNA at 37°C.However, some exceptions apply:
- Some Fermentas restriction enzymes show optimum activity at temperatures other than 37°C. Therefore, the optimum incubation temperature for each restriction enzyme is provided under "Conditions for 100% Activity" in the restriction enzyme description as well as in table "Reaction Conditions for Restriction Enzymes" and in table "Activity of Mesophilic and Thermophilic Enzymes at 37°C".
- Restriction enzymes without recognition sites on lambda DNA are assayed with another specific DNA substrate.
- Restriction enzymes with only a few recognition sites on the lambda DNA are assayed using lambda DNA previously hydrolyzed with another restriction enzyme.
- Restriction enzymes sensitive to Dam or Dcm methylation are assayed with Lambda DNA (dam-, dcm-). For more detailed information regarding methylation effects, see.
Most restriction enzymes are supplied at a user-friendly concentration of 10 u/µl; a number of enzymes are also available at high concentration (HC) - 50 u/µl.
The Labeled Oligonucleotide (LO) test is the most sensitive assay for the purity of restriction enzymes. The assay allows the identification of trace contaminants (endodeoxyribonucleases, exodeoxyribonucleases and phosphatases) in restriction enzyme preparations that are missed by other assays. The 5'-[32P]-labeled synthetic oligonucleotides (single-stranded and double-stranded) used as substrates in the LO test are designed without recognition sites for the restriction enzymes. After these labeled oligonucleotides are incubated with an enzyme, denatured reaction products are separated on a polyacrylamide gel and then analyzed by phospho-imaging. The presence of contaminating other endodeoxyribonucleases or exodeoxyribonuclease results in the degradation of the oligonucleotides (see picture below).
A decrease in the specific radioactivity of the test oligonucleotides indicates the presence of contaminant phosphatases. The restriction enzyme conforms to this quality criterion if there is no degradation of both the single-stranded oligonucleotide and the double-stranded oligonucleotide, and if there is no decrease in band intensity.
Labeled Oligonucleotide (LO) Test.
ss - single-stranded radiolabeled oligonucleotide
ds - double-stranded radiolabeled oligonucleotide
Pure enzyme - Fermentas NotI
Contaminated enzyme - competitor's NotINon-specific Nuclease and Cross-contamination AssayVarying amounts of restriction enzyme (220 units) are incubated for 16 hours with 1 µg of substrate DNA under the recommended assay conditions. After electrophoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. To pass the test, the restriction enzyme must yield an unaltered banding pattern under conditions of up to 160-fold overdigestion (10 units x 16 hours). For information regarding restriction enzyme star activity, see the product description or Certificate of Analysis supplied with each enzyme.
The ligation and recleavage assay tests the integrity of DNA ends. DNA fragments obtained after 2-, 10- and 50-fold overdigestion (units/µg of DNA x hours) are ligated with T4 DNA Ligase and then recut with the same restriction enzyme. Only DNA fragments with intact 5'- and 3'-termini are ligated by T4 DNA ligase, and only molecules with reconstructed recognition sites can then be cleaved by the same restriction enzyme. A restriction enzyme conforms to the quality criterion if the ligation efficiency of DNA fragments, generated by digestion with the restriction enzyme, does not depend on the excess of enzyme used for the initial cleavage of DNA.
The percentage of DNA that can be successfully ligated and then recleaved is presented for each restriction enzyme both in its catalog description and the Certificate of Analysis supplied with each enzyme.The Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 10 units of a restriction enzyme in its optimal buffer. After a 16 hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/Amp agar. An intact lacZ gene will give rise to a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxynucleases, the lacZ reading frame is interrupted, which results in the appearance of white colonies. The higher the ratio of blue to white colonies, the higher the quality of restriction enzyme. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. Details of the assay are given in the Certificate of Analysis of each product. The test is applicable for enzymes recognizing unique sites within the lacZ reporter gene, and for those lacking recognition sites in pUC57. In the latter case, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3'-overhang, 5'-overhang and blunt ends).
All restriction enzymes should be stored at -20°C. For Hin1II, storage at -70°C is recommended.
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 since their quality is not affected by short exposure to +4°C.
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Updated gegužės 09, 2007 15:06
