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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
Topoisomerase I (E. coli) |
 | | | | Topoisomerase I (E. coli) | | | |
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Prices are in US dollars and valid only for US orders.
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- Recognizes non-perfectly matched DNA
- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
Topoisomerase I (E. coli) catalyzes the relaxation of negatively supercoiled DNA. Topoisomerase I has also been implicated in knotting and unknotting DNA (1) and in linking complementary rings of single-stranded DNA into double-stranded rings (2). The intact holoenzyme is a 97 kDa protein.
Source:
A E. coli strain containing the cloned topA gene
Reagents Supplied:
NEBuffer 4
BSA
Properties & Usage
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 0% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Heat Inactivation:
40 units at 65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that catalyzes the relaxation of > 95% of 0.5 μg of pUC19 RF I (negatively supercoiled) DNA in 15 minutes at 37°C in a total reaction volume of 25 μl. DNA supercoiling is assessed by agarose gel electrophoresis in the absence of ethidium bromide.
Concentration:
5,000 units/ml
Storage Conditions:
10 mM Tris-HCl
35 mM (NH4)2SO4
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
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 contaminating exonucleases and endonucleases.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 50 units of Topoisomerase I (E. coli) incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Topoisomerase I (E. coli) with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.2% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 40 units of Topoisomerase I (E. coli) with 1 μg of
pUC19 DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
References
- Liu, L.F., Depew, R.E. and Wang, J.C. (1976) J. Mol. Biol., 106, 439-452.
- Kirkegaard, K. and Wang, J.C. (1978) Nucl. Acids Res., 5, 3811-3820.
Reagents Sold Separately
NEBuffer 4
BSA
Companion Products
DNA Gyrase (E. coli)
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