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Exonuclease I, E.coli

#EN0581 Supplied with: 10X Reaction Buffer	4000 u (20 u/µl) 1 ml
#EN0582 Supplied with: 10X Reaction Buffer	20,000 u (20 u/µl) 5x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EN0581, #EN0582 MSDS (English) MSDS (English-USA) MSDS (German)

 

Feature
Active in Fermentas buffers for PCR (see table).

Description
The Exonuclease I (ExoI) degrades single-stranded DNA in a 3'=>5' direction, releasing deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaving 5'-terminal dinucleotides intact. It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups (1).

Source
E.coli cells with a cloned E.coli sbcB gene.

Molecular Weight
54.5 kDa monomer.

Applications

• Purification of PCR products:
  • for direct sequencing, see Protocol for PCR Product Clean-up (procedure according (2));
  • in one-tube “megaprimer” PCR mutagenesis (3).
• Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures.
• Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus (4).

Quality Control
The absence of endodeoxyribonucleases, double-stranded exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.

Concentration
20 u/µl

Definition of Activity Unit
One unit of the enzyme catalyzes the release of 10 nmol of acid soluble nucleotides in 30 min at 37°C.
Enzyme activity is assayed in the following mixture: 67 mM glycine-KOH (pH 9.5), 6.7 mM MgCl2, 1 mM DTT and 0.17 mg/ml single-stranded [³H]-DNA.

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

10X Reaction Buffer
670 mM glycine-KOH (pH 9.5 at 25°C), 67 mM MgCl₂, 10 mM DTT.

Inhibition and Inactivation
Inactivated by heating at 80°C for 15 min.

Related Products

Endonuclease IV, E.coli Water, nuclease-free

References

1. Lehman, I.R., Nussbaum, A.L., The deoxyribonucleases of Escherichia coli. V. On the specificity of exonuclease I (phosphodiesterase), J. Biol. Chem., 239, 2628-2636, 1964.
2. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.
3. Nabavi S., Nazar R.N., Simplified one-tube “megaprimer” polymerase chain reaction mutagenesis, Anal Biochem., 2005, 2, 346-348, 2005.

4. Rosamond, J., et al., Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+, J. Biol. Chem., 254, 8646-8652, 1979.

Updated kovo 18, 2008 09:39