T4 Endonuclease VII

Description

Description:
Endonuclease VII is the product of gene 49 of bacteriophage T4. It has a mass of 18 kDa. T4 Endonuclease VII involves in DNA-packaging, genetic recombination and mismatch repair in vivo. It has also been demonstrated in vitro to resolve single-base misparings, heteroduplex loops and branched DNAs, such as four-way Holliday junctions and three-way Y-structures.

Source:
A recombinant E. coli strain carrying the cloned T4 Endonuclease VII gene

Unit Definition:
One unit (0.5 ng) of the enzyme resolves 50% of 1 pmol FAM labeled 28mer oligonucleotide substrate [1] within the immobile 4-way Holliday junction structure in 30 minutes at 37°C in 50mM Tris-HCl, pH 8.0, 10mM MgCl2, 10mM 2-ME and 0.1 μg/μl BSA.

Specific Activity:
2000 U/µg

Recommended Storage Condition:
 -20°C

Experimental Data:

Figure1.  Performance of MCLAB’s T4 Endonuclease VII analyzed by capillary electrophoresis. (a) Negative control sample analysis, 10 pmol of FAM labeled 28mer oligonucleotide substrate. (b) 40 U of MCLAB’s T4 Endonuclease VII was able to fully resolve 10 pmol of FAM labeled 28mer oligonucleotide substrate with a 4-way Holliday junction structure (30 minutes at 37°C in 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM 2-ME and 0.1 μg/μl BSA).

Figure2.  MCLAB’s T4 Endonuclease VII and T7 Endonuclease I (from other supplier) activity comparison.  (a) 10 U (275 fmol) of MCLAB T4’s Endonuclease VII can resolve 64% of 10 pmol of 4-way junction substrate (enzyme to substrate molar ratio 1:70) in 30 minutes at 37°C in 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM 2-ME and 0.1 μg/μl BSA. (b) 40 U (368 fmol) of T7 Endonuclease I (supplier N) only resolves 43% of 10 pmol of 4-way junction substrate (enzyme to substrate molar ratio 1:50) in 30 minutes at 37°C in 50 mM NaCl, 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM DTT. Compared to T7 Endonuclease I (supplier N), MCLAB’s T4 Endonuclease VII resolves 4-way Holliday Junctions at a higher yield in 30 minutes with less enzyme.

T4 Endonuclease VII Protocol

For 4-way junction substrate:

1.       Prepare the following reaction:

2.       Mix well and incubate the reaction @ 37°C for 30min.

3.       Use 1 µl reaction to analyze the cleaved fragments on Capillary electrophoresis.

For single base mismatch substrate:

1.       Use about 200-400ng DNA fragment that contains single base mismatch for each reaction:

2.      Mix well and incubate the reaction @ 37°C for 30min.

3.      Run a 2% agarose gel and check for cleaved bands.
Table. Comparison of T4 Endonuclease VII and T7 Endonuclease I

* See Figure 2 for T4 endonuclease VII and T7 endonuclease I activity comparison result.

Supplied in:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C

Supplied With:
10x T4 Endonuclease VII Reaction Buffer:
500 mM Tris-HCl
100 mM MgCl2
100 mM 2-mercaptoethanol
1 mg/ml BSA
pH 8.0 @ 25°C

Reference:
1. Golz, S., Birkenbihl, R. P., and Kemper, B. (1995), DNA Research 2, 277-284.

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