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BitNuclease (Benzonase® Alternative, Purity > 98%)
Description
BitNuclease is a universal endonuclease that is genetically
engineered from Serratia marcescens extracellular endonuclease. It
digests both single and double stranded RNA and DNA (in either
linear, circular or super coiled form) into 3-5 base fragments, which is
below the hybridization limit. BitNuclease has high activity and broad
substrate tolerance, making it an ideal enzyme for removal of nucleic
acids during protein preparation. It can be added along with cell lysis
buffer to eliminate viscosity and increase yields of proteins, including
nuclear proteins.
Components
Contents
Cat#:B16002
Cat#:B16003
Cat#:B16012
BitNuclease Purity > 98%
25KU (250U/ul)
250KU (250U/ul)
25KU (25U/ul)
Unit definition
One enzymatic unit will digest salmon sperm DNA to a ∆A260 of 1.0
(corresponding to degradation of 37µg DNA) in 30 min at pH 8.0 at
37°C.
Purity > 98% (SDS-PAGE), endotoxin < 0.22 EU/mg, no protease
activity detected.
Storage
This product can be stored at -20°C for 2 years, or 4°C for 2 months.
Should be prevented from freezing.
Storage/dilution buffer formula: 50mM Tris-HCl, pH 8.0, 20mM NaCl,
5mM MgCl2, 50% Glycerol.
Application
25U/mL
4°C 30-60min
Nuclear protein production
100U/mL
37°C 10min or 4°C 30min
Virus Purification
12.5U/mL
37°C 30min
Vaccine Production
0.9-1.1U/mL
30-37°C 4-8 hrs
Protein Purification
Troubleshooting
Problem
Suggestion(s)
In different application
processes, when should
I introduce BitNuclease?
To achieve the best nucleic acid removal effect,
BitNuclease should be added at the same time
when the targeted nucleic acids were released.
At the same time, high salt concentration and
other conditions in which BitNuclease
activity is inhibited should be avoided.
At low temperatures,
how much more
BitNuclease do
I need to add?
BitNuclease activity decreases at temperatures below
37°C. Adding more BitNuclease (2-5X volume) or
giving a longer incubation time (2-3 hours)
can compensate for this decrease.
Is BitNuclease stable?
Is it OK after being
left out on the
bench several days?
BitNuclease is extremely stable.
After extended
incubations at 37 °C for
2 weeks, BitNuclease maintained >90% activity.
Why isn’t
BitNuclease working?
BitNuclease will reach its optimum activity in the
presence of Mg2+. The activity is inhibited by
monovalent cation concentrations >300 mM,
phosphate concentrations >100 mM,
and by ammonium sulfate concentrations >100 mM.
In addition, concentrations of >1 mM EDTA
will also inhibit Benzonase® endonuclease activity.
How can I inhibit
BitNuclease activity?
Chelating agents like EDTA, cause loss of free Mg2+,
which subsequently inhibit BitNuclease activity.
1mM EDTA can be used in this purpose. The inhibition
status can be reversed by adding more MgCl2.
How to remove
BitNuclease?
Removal of BitNuclease can be accomplished by
downstream protein purification procedure. Up to
97% of BitNuclease is removed by anion
exchange or gel filtration chromatography.
Notice
BitNuclease requires 1-2mM Mg2+ for activation, please make sure
Mg2+ is added in the solution.
1mM or higher EDTA inhibits BitNuclease activity serverely.
BitNuclease is functional between pH 6 and 10, with best efficiency in
pH 8-8.5. Functional temperature is from 0°C to 42°C and optimal
temperature is 37°C.
BitNuclease can tolerate buffers containing up to 150mM monovalent
cation, 100mM phosphate, 100mM ammonium sulfate, 100mM
Guanidine HCl, 0.1% SDS, 1% Triton X-100, 1% Tween 20. This is
compatible with most buffers used in protein purification.
Are there safety concerns
for BitNuclease?
Is BitNuclease free
of protease activity?
Protocol
BitNuclease is a “ready to use” product; however different amounts
are used according to different applications. Suggested
concentrations are listed below. The concentration of nucleic acid in
specific samples can vary, so it may be necessary to adjust the
volume of BitNuclease to the requirements of different situations.
Procedure
Concentration
Is BitNuclease
compatible with protease
inhibitor cocktails?
Toxicological studies with BitNuclease showed that
no toxic effects were observed, even at very high
doses. Furthermore, no mutagenic potential
was observed in vivo, even at high dosage.
Yes, BitNuclease is verified to have no detectable
protease activity. However, if protease is
present in the sample
itself, BitNuclease will be be degraded.
BitNuclease is compatible with most components in
protease inhibitor cocktails, except EDTA.
Concentrations of 1 mM or higher
EDTA will inhibit the activity of BitNuclease.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]