Enzyme Quality

All Thermo Scientific 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 Thermo Scientific product line meets 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 Product Information insert 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 Thermo Scientific consistently guarantee the highest quality of DNA/RNA modifying enzymes and other proteins.

Activity Assays

Activity unit definitions for Thermo Scientific 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.

Labeled Oligonucleotide Test (LO)

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, see Table 1, 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, see Fig. 1 below.

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

Double-stranded Endodeoxyribonuclease Assay (dsEndo)

The assay is performed in 50µl of reaction mixture containing reaction buffer, enzyme and 1µg of covalently closed circular (supercoiled) DNA (either pUC19 DNA or phiX174 RF1 DNA). After incubation under the appropriate conditions, see Table 1, 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 50µl of reaction mixture containing reaction buffer, enzyme and 1µg of covalently closed circular single-stranded DNA of M13mp19. After incubation under appropriate conditions, see Table 1, 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 50µl of reaction mixture containing reaction buffer, enzyme and 1µg of sonicated [³H]-labeled DNA from E.coli. After incubation under the appropriate conditions, see Table 1, 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 50µl of reaction mixture containing reaction buffer, enzyme and 1µg of either the lambda DNA or plasmid DNA fragments. After incubation under appropriate conditions, see Table 1, the DNA is analyzed on an agarose gel. The product passes this quality control test if DNA fragments are not degraded.

Ribonuclease Assay (RNase)

The assay is performed in 50µl of reaction mixture containing reaction buffer, enzyme and 1µg of [³H]-labeled RNA. After incubation under the appropriate conditions, see Table 1, the RNA is precipitated with trichloroacetic acid and the radioactivity of the supernatant is determined. Ribonuclease activity is expressed as a percent of the total RNA radioactivity released into the acid soluble fraction. Details of the assay are provided in the Product Information insert supplied with each product.

Protease Assay

The assay is performed in 200µl of reaction mixture containing 10mM Tris-HCl buffer (pH7.5), enzyme and 200µg of azocasein. After incubation under the appropriate conditions, see Table 1, the reaction is terminated with trichloroacetic acid and the absorbance of the supernatant is measured at 400nm. The product passes this quality control test if azocasein is not degraded.

Functional Assays

Assays performed for a particular modifying enzyme or other protein are indicated both in the product entry and in the Product Information insert supplied with each product.