Source: An E. coli strain that carries a TaqαI overproducing plasmid (F. Barany using an NEB clone). TaqαI has a two amino acid replacement at its amino terminus. This allows for a higher level of expression without interfering with its catalytic properites.
Reagents Supplied: NEBuffer 4 (10X)
BSA (100X)
Enzyme Properties Activity in NEBuffers:
NEBuffer 1:
50%
NEBuffer 2:
75%
NEBuffer 3:
100%
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.
Methylation Sensitivity:
dam methylation: Blocked by overlapping
dcm methylation: Not sensitive
CpG methylation: Not sensitive
Activity at 37°C: 10%
Heat Inactivation: 80°C for 20 minutes
Survival in a Reaction: Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 4 Supplemented with 100 μg/ml Bovine Serum Albumin Incubate at
65°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 required to digest 1 μg of λ DNA in 1 hour at 65°C in a total reaction volume of 50 μl.
Concentration: 20,000 units/ml and 100,000 units/ml
Unit Assay Substrate: λ DNA
Storage Conditions: 10 mM Tris-HCl 300 mM KCl 1 mM Dithiothreitol 1 mM EDTA 500 µg/ml BSA 50% Glycerol
pH 7.5 @ 25°C
Storage Temperature: -20°C
Diluent Compatibility: Diluent B
Notes General notes:
TaqαI has a two amino acid replacement at its amino terminus. This allows for a higher level of expression without interfering with its catalytic properites.
Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 80-fold overdigestion with TaqαI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with TaqαI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 300 units of TaqαI incubated for 16 hours at 65ºC
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 500 units of TaqαI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 65ºC
released < 0.15% of the total radioactivity.
