What is the appropriate buffer for EcoRI?
What is the appropriate buffer for EcoRI?
Thermo Scientific 10X Buffer EcoRI is the optimal buffer recommended for use with EcoRI restriction enzyme and is premixed with BSA for enhanced stability.
What is the composition of the buffer for the restriction enzyme HindIII?
Storage Buffer HindIII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/mL BSA and 50% glycerol.
What bacteria does HindIII come from?
HindIII (pronounced “Hin D Three”) is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis.
What does EcoRI digestion produce?
EcoRI creates 4 nucleotide sticky ends with 5′ end overhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G↓AATTC, which has a palindromic, complementary sequence of CTTAA↓G.
What is digest buffer?
Differex™ Digestion Buffer is used in differential extractions of samples that contain a mixture of epithelial and sperm cells. A digestion combination of this buffer and Proteinase K can be used to selectively lyse epithelial cells while leaving sperm cells intact.
Why do EcoRI and HindIII require two-step digestion?
Since EcoRI and HindIII use different buffer, to have efficient cleavage for both of them and avoid star activity due to a change in reaction conditions, a sequential digestion is recommended. NEB and other companies have enzyme versions with reduced star activity to avoid this two-step digestion.
What buffers are available for double digesting enzymes?
Setting up a Double Digestion with a Unique Buffer (designated “U”) NEB currently supplies three enzymes with unique buffers: EcoRI, SspI and DpnII. In most cases, DpnII requires a sequential digest. Note that EcoRI has an HF version which is supplied with CutSmart Buffer.
Why do restriction enzymes digest EcoR1 and HindIII in sequential order?
In NEB systems they prefer a double digestion of EcoR1 and HindIII to be sequential (one after the other). Because some restriction enzymes, particularly EcoRI, show star activity (they cleave in sequences that differ from their recognition site) when the reaction conditions are not optimal.
How do I set up a Double Digest in cutsmart buffer?
Setting up a Double Digestion. Double digests with NEB’s restriction enzymes can be set up in CutSmart Buffer. Otherwise, choose an NEBuffer that results in the most activity for both enzymes. If star activity is a concern, consider using one of our High Fidelity (HF®) enzymes. Set up reaction according to recommended protocol.
