The type IIP restriction enzyme NotI from Nocardia otitidis-caviarum is a homodimer which recognizes the 8 basepair DNA sequence 5′- GC/GGCCGC -3′ and cleaves both strands of DNA to create 5′, 4-base cohesive overhangs (Qiang and Schildkraut, 1987).
What are HF enzymes?
High Fidelity Restriction Enzymes have been engineered by exchanging functional amino acid residues and then screening for optimal mutants that perform under a wide range of conditions.
Is NotI a restriction enzyme?
Thermo Scientific NotI restriction enzyme recognizes GC^GGCCGC sites and cuts best at 37°C in O buffer (Isoschizomers: CciNI). 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.
What is the restriction site for ecor1?
Key Features of EcoRI It is a type II restriction enzyme, which cuts specifically at the restriction site having 5′-GAATTC-3′ sequence. It cuts the DNA at a specific site forming sticky ends. Restriction site of EcoRI is a palindrome and it cuts DNA after G forming sticky ends with AATT.
Is Taq polymerase High fidelity?
One unit of Platinum® Taq DNA Polymerase, High Fidelity, is the amount of enzyme required to incorporate 10 nmoles of deoxyribonucleotide into DNA in 30 min at 74°C.
What is a high fidelity polymerase?
The fidelity of a polymerase refers to its ability to insert the correct base during PCR. High-fidelity PCR, utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest.
Why is it called star activity?
It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed “”star”” activity.
How can we stop star activity?
If you are concerned about star activity, we recommend the following guidelines. 1. Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction.
What is the difference between Noti-HF and Neb?
NotI-HF, NEB has the same specificity as NotI, but has been engineered for significantly reduced star activity and performance in a single buffer ( CutSmart ® Buffer ). All HF-restriction enzymes come with Gel Loading Dye, Purple (6X) .
Why HF restriction enzymes?
HF enzymes are all Time-Saver qualified and can therefore cut substrate DNA in 5-15 with the flexibility to digest overnight without degradation to DNA. Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design.
What is the resolution of Noti-HF®?
The resolution is at the single nucleotide level. One unit is defined as the amount of enzyme required to digest 1 μg of pBC4 DNA in 1 hour at 37°C in a total reaction volume of 50 μl. NotI-HF® has the same specificity of NotI (NEB #R0189), but it has been engineered for reduced star activity.
Does Noti have a high fidelity (HF) version?
NotI has a High Fidelity version NotI-HF ® (NEB #R3189). High Fidelity (HF) Restriction Enzymes have 100% activity in rCutSmart Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity.
