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DNA Modifying Enzymes and Cloning >
Nucleases >
RecJf |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
- 5' → 3' exonuclease
Description:
RecJf is a single-stranded DNA specific exonuclease that catalyzes the removal of deoxy-nucleotide monophosphates from DNA in the 5' → 3' direction (1). RecJf is a recombinant fusion protein of RecJ and maltose binding protein (MBP). It has the same enzymatic properties as wild-type RecJ. Fusion to MBP enhances RecJf solubility (2).
When DNA containing a 22 base 5' extension is used as a substrate for RecJf, the resulting products are a mixture of DNA fragments that have blunt-ends, 5' extensions (1-5 nucleotides) and recessed 5' ends (1-8 nucleotides) (3). RecJf does not require a 5' phosphate (3).
Source:
RecJf is overexpressed and purified as a C-terminal fusion to maltose-binding protein (MBP). MBP does not affect the catalytic activity of RecJf but does enhance RecJf solubility (2).
Reagents Supplied:
NEBuffer 2 (10X)
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to produce 0.05 nmol TCA soluble deoxyribonucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X NEBuffer 2 with 1.5 µg sonicated single-stranded [3H]-labeled E. coli DNA.
Concentration:
30,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
FAQs
- What is the activity of RecJf in the NEBuffers?
- Can DNA be blunted using RecJf?
- What is the difference between RecJf and Exonuclease I (NEB# M0293)?
- Can RecJf be heat inactivated?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of endonucleases and contaminating exonucleases.
3' to 5' Exonuclease Activity:
No detectable 3' → 5' nuclease activity was observed when 30 units
of RecJf was incubated with substrates containing either 3' extensions or blunt ends.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of RecJf with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Single-stranded Endonuclease Assay::
Incubation of 50 units of RecJf with 1 µg of ΦX174 Virion DNA for 4 hours at 37°C in a 50 µl reaction resulted in no decrease in the amount of closed circular DNA as determined by agarose gel electrophoresis.
References
- Lovett, S.T., Kolodner, R.D. (1989) Proc. Natl. Acad. Sci. USA, 86, 2627-2631.
- Lovett, S. and Whitaker, R., unpublished observations.
- Whitaker, R., unpublished observations.
Reagents Sold Separately
NEBuffer 2
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