Why do you add the enzyme last?
The restriction enzyme is usually the last component added to a reaction to ensure that it is not exposed to extreme conditions. When many similar digests are being prepared, it may be convenient to create premixes of common reagents. Mix all solutions containing restriction enzymes gently to avoid enzyme inactivation.
What is Tango buffer?
Tango buffer has been designed for double digestions of DNA with conventional restriction enzymes. To ensure consistent enzyme performance, Thermo Scientific restriction enzyme buffers contain BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.
Why is bamh1 used?
BamHI (from Bacillus amyloli) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 b.p.) of DNA and specifically cleaving them at a target site. This exhibit focuses on the structure-function relations of BamHI as described by Newman, et al.
What can affect restriction enzyme digestion?
Factors that affect Restriction Enzyme Activity
- Temperature: Most endonucleases digest the target DNA at 37 °C with few exceptions.
- Cofactors: Restriction endonucleases require certain cofactors or combination of cofactors to digest at the recognition site.
What is Ne buffer?
NEB’s restriction enzyme buffer system makes your restriction digests easy and convenient. We are able to offer >215 restriction enzymes that cut in a single buffer, rCutSmart™ . This improves ease-of-use, especially when performing double digests.
How big is one unit of bspei enzyme?
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam-) in 1 hour at 37°C in a total reaction volume of 50 µl. BspEI is an isoschizomer of BspMII. Blocked by overlapping dam methylation and impaired by CpG methylation.
What are the reaction conditions for kpn2i ( bspei )?
Thermo Scientific Kpn2I (BspEI) restriction enzyme recognizes T^CCGGA sites and cuts best at 55°C in Tango buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.
How are nonspecific inhibitors of enzymes irreversible?
Nonspecific Inhibitors: A nonspecific inhibition effects all enzymes in the same way. Non-specific methods of inhibition include any physical or chemical changes which ultimately denaturesthe protein portion of the enzyme and are therefore irreversible.
Which is E coli strain carries the bspei gene?
A E. coli strain that carries the BspEI gene from Bacillus species (H. Kong). One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam-) in 1 hour at 37°C in a total reaction volume of 50 µl.