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 Assays

Activity 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.

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 – Fermentas NotI
Contaminated enzyme – competitor's NotI

Double-stranded Endodeoxyribonuclease Assay (dsEndo)

The assay is performed in 50 µl of reaction mixture containing reaction buffer, enzyme and 1 µg 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 50 µl of reaction mixture containing reaction buffer, enzyme and 1 µg 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 50 µl of reaction mixture containing reaction buffer, enzyme and 1 µg 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 50 µl of reaction mixture containing reaction buffer, enzyme and 1 µg 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.

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, see Table 1. 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 and/or terminal transferases, 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 50 µl of reaction mixture containing reaction buffer, enzyme and 1 µg 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 Certificate of Analysis supplied with each product.

Protease Assay

The assay is performed in 200 µl of reaction mixture containing 10 mM Tris-HCl buffer (pH 7.5), enzyme and 200 µg 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 400 nm. 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 Certificate of Analysis supplied with each product.

 

Table 1. Conditions for Quality Control Assays.

Enzyme	Amount per assay	Incubation temp., °C	Incubation time, hour	QC performed
T4 DNA Ligase	200 u	37	4	dsEndo, LO, B/W, RNase
T4 RNA Ligase	50 u	37	4	dsEndo, LO, RNase
FastAP™ Thermosensitive Alkaline Phosphatase	10 u	37	4	dsEndo, Exo I, RNase
Shrimp Alkaline Phosphatase (SAP)	10 u	37	1	dsEndo, Exo I, B/W, RNase
T4 Polynucleotide Kinase (T4 PNK)	50 u	37	4	dsEndo, Exo I, RNase
DreamTaq™ DNA Polymerase	10 u	37	4	dsEndo, Exo II, RNase
Maxima® Hot Start Taq DNA Polymerase	10 u 10 u	37, 65 37	4 4	Exo I dsEndo, RNase
TrueStart™ Hot Start Taq DNA Polymerase	10 u 10 u	37, 70 37	4 4	Exo II, dsEndo RNase
Taq DNA Polymerase (recombinant)	10 u	37, 70	4	dsEndo, Exo II, RNase
Taq DNA Polymerase (native, without BSA) (native, with BSA)	10 u 10 u	70 70	4 4	dsEndo, Exo II, RNase
Pfu DNA Polymerase (native)	10 u	72	4	dsEndo, Exo II
Pfu DNA Polymerase (recombinant)	10 u	37, 72	4	dsEndo, Exo II
phi29 DNA Polymerase	100 u	30	4	ds-, ssEndo
DNA Polymerase I, E.coli	20 u	37	4	dsEndo
Klenow Fragment	20 u	37	4	dsEndo
Klenow Fragment, exo-	20 u	37	4	dsEndo, Exo I, LO
T4 DNA Polymerase	10 u	37	4	ds-, ssEndo
T7 DNA Polymerase	10 u	37	4	dsEndo
Terminal Deoxynucleotidyl Transferase (TdT)	60 u	37	4	dsEndo, LO, RNase
RevertAid™ H Minus M-MuLV Reverse Transcriptase	2000 u	37	4	dsEndo, LO, RNase
RevertAid™ M-MuLV Reverse Transcriptase	2000 u	37	4	dsEndo, LO, RNase
M-MuLV Reverse Transcriptase	200 u	37	4	dsEndo, LO, RNase
AMV Reverse Transcriptase	25 u 40 u	37 37	16 4	dsEndo Exo I, RNase
T7 RNA Polymerase	200 u	37	1	dsEndo, Exo I, RNase
SP6 RNA Polymerase	200 u	37	1	dsEndo, Exo I, RNase
T3 RNA Polymerase	200 u	37	1	dsEndo, Exo I, RNase
RiboLock™ RNase Inhibitor	200 u	37	4	dsEndo, LO, RNase
DNase I, RNase-free	10 u	37	4	RNase, Protease
Endonuclease IV, E.coli (Endo IV)	20 u	37	1	dsEndo, Exo I, RNase
Endonuclease V, T.maritima (Endo V)	25 u	37	4	dsEndo, Exo I, LO, RNase
Exonuclease I, E.coli (Exo I)	100 u	37	16	ds-, ssEndo, Exo II, RNase
Exonuclease III, E.coli (Exo III)	25 u	37	4	dsEndo
Lambda Exonuclease	100 u	37	4	dsEndo
RNase A, DNase and protease-free	5 µg 25 µg	37 37	18 18	dsEndo, LO Protease
RNase T1	1000 u 10000 u	37 37	18 18	dsEndo, LO Protease
RNase A/T1 Mix	2 µl 10 µl	37 37	18 18	dsEndo, LO Protease
RNase I, E.coli	80 u	37	4	dsEndo, Exo I, LO
RNase H, E.coli	10 u	37	1	dsEndo, Exo I, RNase
Agarase	5 u	37	4	dsEndo, LO, B/W, RNase
Proteinase K (recombinant), PCR grade	200 µg 40 µg	37 37	16 16	dsEndo, Exo I, RNase
Pyrophosphatase, Inorganic (from yeast)	1 u	37	24	dsEndo, Exo I, LO, RNase
Uracil-DNA Glycosylase (UDG)	50 u	37	4	dsEndo, LO, RNase