What happens if you add too much restriction enzyme?
What happens if you add too much restriction enzyme?
Incomplete digestion is a frequently encountered issue when using restriction endonucleases. Incomplete digestion may occur when too much or too little enzyme is used. The presence of contaminants in the DNA sample can inhibit the enzymes, also resulting in incomplete digestion.
Why we use sequential digestion during double digestion of DNA?
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. Two enzyme selected for digestion may have different buffering conditions thats why enzymes are supplied with specific buffers.
What affects restriction enzyme digestion?
The digestion activity of restriction enzymes depends on the following factors: 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 wrong with HindIII and KpnI on pcdna3?
I realized that HindIII and KpnI are right next to each other on pcDNA3.0. Therefore, the problem might be that either one or both enzymes cannot cut (sterics). Would performing single digestions followed by purification for each enzyme be the way to go?
How do I set up Double Digests with Neb’s restriction enzymes?
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.
What is the best temperature for HindIII restriction enzymes?
Thermo Scientific HindIII restriction enzyme recognizes A^AGCTT sites and cuts best at 37°C in R 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.
What is the high fidelity version of KpnI?
KpnI has a High Fidelity version KpnI-HF ® ( NEB #R3142 ). High Fidelity (HF) Restriction Enzymes have 100% activity in CutSmart Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity.
