By Where does NdeI cut?
Description. Thermo Scientific NdeI restriction enzyme recognizes CA^TATG sites and cuts best at 37°C in O buffer (Isoschizomers: FauNDI).
What are the restriction site sequences for NdeI?
NdeI: a restriction endonuclease from Neisseria denitrificans which cleaves DNA at 5′-CATATG-3′ sequences.
How can we prevent restriction digestion?
Restriction Enzyme Digest Protocol
- Add components to a clean tube in the order shown:
- Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.
- Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA.
What is the recognition sequence for EcoRI?
EcoRI recognizes the sequence GAATTC, and cuts both DNA strands between the G and the A nucleotides. Protruding from the cut ends will be single-stranded DNA “tails” having the sequences AATT.
What is the restriction site for HindIII?
1. The restriction enzyme HindIII cuts DNA at the sequence AAGCTT, and the restriction enzyme HpaII cuts DNA at the sequence CCGG.
How many types of restriction enzymes are there?
four types
Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors.
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.
How do you know if your restriction digestion was successful?
If the digested product would be visible at a lower coordinate on the gel, it would have made things easy. You can amplify your digested fragment with primer beginning in the flankers region and with only 3-4 bp in the intern 8680 bp region. If you do not get PCR fradments, was the digestion successfully.
How do you do double restriction digestion?
Setting up a Double Digestion In most cases, double digests with NEB’s restriction enzymes can be set up in rCutSmart 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.
What is EcoRI and HindIII?
Description. Thermo Scientific Lambda DNA/EcoRI+HindIII Marker is recommended for sizing of linear double-stranded large DNA fragments in agarose gels. Lambda DNA is digested to completion with the appropriate Thermo Scientific restriction enzyme(s) and purified and dissolved in storage buffer.