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Results: 4

1.
Figure 4.

Figure 4. From: The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

Star activity is site-specific. While a clear star activity band is seen for NotI when pXba is used as the substrate, no star activity is observed when pBC4 is used. Asterisk is a star activity band, and hash is a partial activity band in every panel. NotI is 2-fold serially diluted with diluent C. The reaction contains 3 μl of diluted NotI, 1.2 μl pXba or pBC4 (0.6 μg), 3 μl buffer N2 and 22.8 μl of H2O. Reaction is at 37°C for 1 h. Upper panel: Lane 1: 4800 U NotI; lane 8: 38.4 U NotI (HNS); lane 14: 0.6 U NotI (LCC); lane 20: 0.01 U; FI = 64. Bottom Panel: Lane 1: 2400 U NotI; lane 13: 0.6 U NotI (LCC); lane 20: 0.005 U; FI ≥ 4000-.

Hua Wei, et al. Nucleic Acids Res. 2008 May;36(9):e50-e50.
2.
Figure 3.

Figure 3. From: The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

Representative example of the ‘Variable’ class of REase. Comparison of FIs of NotI in buffer N4 (A) and buffer N3 (B) using pXba DNA as the substrate. NotI is 2-fold serially diluted with diluent C. The reaction contains 3 μl of diluted NotI, 1.2 μl pXba DNA (0.6 μg), 3 μl buffer N4 (A) or buffer N3 (B) and 22.8 μl of H2O. The reaction is at 37°C for 1 h. Asterisk is a star activity band, and ‘hash’ is a partial activity band. (A) Lane 1: 4800 U NotI; lane 10: 9.6 U NotI (HNS); lane 14: 0.6 U NotI (LCC); lane 20: 0.01 U, NotI; FI = 16. (B) Lane 1: 19 200 U; lane 7: 300 U (HNS); lane 16: 0.6 U (LCC); lane 20: 0.04 U. FI = 500.

Hua Wei, et al. Nucleic Acids Res. 2008 May;36(9):e50-e50.
3.
Figure 2.

Figure 2. From: The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

Representative examples of the ‘Excellent’ and ‘Good’ classes of REases in terms of star activity. (A) REase in the ‘Excellent’ class. Cleavage of pBR322 by MspI in buffer N1: MspI is 2-fold serially diluted with diluent A. The reaction contains 3 μl of diluted MspI, 0.6 μl pBR322(0.6 μg), 3 μl buffer N1 and 23.4 μl of H2O. Reaction is at 37°C for 1 h. Lane 1: 9600 U MspI; lane 15: 0.6 U MspI (LCC); lane 20: 0.02 U MspI. FI ≥ 16 000. ‘Hash'is a partial activity band, and there is no star activity band. (B) REase in ‘Good’ Class. Cleavage of λ DNA by BstBI in buffer N2. BstBI is 2-fold serially diluted with diluent A. The reaction contains 3 μl of diluted BstBI, 1.2 μl λ DNA (0.6 μg), 3 μl buffer N2 and 22.8 μl of H2O. The reaction is at 65°C for 1 h. Lane 1: 2400 U BstBI; lane 5: 150 U BstBI (HNS); lane 13: 0.6 U BstBI (LCC); lane 20: 0.005 U BstBI, FI = 250. Asterisk is a star activity band, and ‘hash’ is a partial activity band.

Hua Wei, et al. Nucleic Acids Res. 2008 May;36(9):e50-e50.
4.
Figure 1.

Figure 1. From: The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

Definition of fidelity index. λ DNA is cleaved by ScaI in buffer N3. The enzyme concentration is reduced by a series of two fold dilutions in diluent A. Lane1: 0.5 μg NEB 1-kb DNA Marker; lane 2 contains 600 U; lane 10 contains 2.4 U; lane 12 contains 0.6 U; lane 21 contains 0.002 U ScaI. Reactions contain 3 μl of diluted ScaI, 1.2 μl λDNA (0.6 μg), 3 μl buffer N3 and 22.8 μl of H2O. The reaction is at 37°C for 1 h. The vertical arrows correspond to the two critical points: HNS—the Highest REases amount showing No Star activity and LCC—the Lowest REase amount needed for Complete Cleavage on cognate sites. FI = HNS/LCC, it is 4 in this case. Asterisk is a star activity band, and ‘hash’ is a partial activity band. This is also a representative example of the ‘Star-prone’ class REases.

Hua Wei, et al. Nucleic Acids Res. 2008 May;36(9):e50-e50.

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