PureExtreme® Quality

FastDigest® restriction enzymes are produced under controlled environment conditions and tested in all industry-standard quality control assays. They are also tested in our unique Labeled Oligonucleotide (LO) test, which is the most sensitive test for the detection of trace activities of endo-, exodeoxyribonucleases and phosphatases.
The PureExtreme® Quality of FastDigest® restriction enzymes makes them the enzymes of choice for even the most demanding applications.

Activity Assay

In general, enzymes are assayed with lambda phage DNA at 37°C. However, some exceptions apply:

• The few thermophilic FastDigest® enzymes (BseNI, TaaI, TaiI, TaqI, Tru1I, Tsp509I (TasI), TspRI (TscAI)) are assayed at 65°C.
• FastDigest® enzymes that do not have recognition sites on lambda DNA are assayed with other specific DNA substrates, as indicated in the Certificates of Analysis.
• FastDigest® enzymes with only a few recognition sites on the lambda DNA are assayed using lambda DNA cut with another restriction enzyme.
• FastDigest® sensitive to Dam or Dcm methylation are assayed using DNA purified from dam-, dcm- strain of E.coli.

A detailed description of the activity assay for each enzyme is listed in product descriptions and Certificates of Analysis.

Quality Control

 Labeled Oligonucleotide (LO) Test

1µl of FastDigest® enzyme is incubated with 5'-[³²P]-labeled single-stranded and double-stranded synthetic oligonucleotides in 1X FastDigest® buffer for 1 hour at the recommended temperature. The restriction enzyme passes this quality control test if there is no degradation of labeled oligonucleotides and no decrease in their specific radioactivity.

Figure 1. Labeled Oligonucleotide (LO) Test.
ss – single-stranded radiolabeled oligonucleotide
ds – double-stranded radiolabeled oligonucleotide
Pure enzyme – Fermentas NotI
Contaminated enzyme – competitor's NotI

Prolonged Incubation / Star Activity Assay

The star activity assay confirms that no alterations in the DNA digestion pattern occur after prolonged incubation with FastDigest® enzymes.
1 µl of FastDigest® enzyme is incubated with 1 µg of substrate DNA at the recommended temperature for up to 16 hours. After electrophoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. The maximal incubation time that does not cause any star activity is indicated for each FastDigest® enzyme in its Certificate of Analysis.

Ligation and Recleavage Assay

The ligation and recleavage assay confirms the integrity of DNA ends. DNA fragments obtained after overdigestion with 1 µl of FastDigest® enzyme for 1 hour are ligated with T4 DNA ligase and then recut with the same enzyme. A FastDigest® restriction enzyme conforms to the quality criterion if the observed ligation efficiency is identical to that of conventional enzyme.
The percentage of DNA that can be successfully ligated and then recleaved for each FastDigest® restriction enzyme is indicated in the Certificate of Analysis.

Blue/White (B/W) Cloning Assay

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 1 µl of a FastDigest® restriction enzyme in 1X FastDigest® buffer. After a 1 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. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. For FastDigest® restriction enzymes lacking recognition sites in pUC57 DNA, 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).