What is double digestion with restriction enzymes?
What is double digestion with restriction enzymes?
A double digest is one where two restriction enzymes are used to digest DNA in a single reaction. In this case you will be using EcoR I and BamH I. There is only one site in the plasmid vector for each of these enzymes and they are located on either side of your insert DNA.
What is a double digest electrophoresis?
Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration.
How do restriction enzymes digest DNA?
Protocol for DNA Digestion with Two Restriction Enzymes 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. The digested DNA is ready for use in research applications.
Why are two restriction enzymes used in gel electrophoresis?
These enzymes cut both strand of the target DNA at different spots creating 3′- or 5′-overhangs of 1 to 4 nucleotides (so-called sticky ends). To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction enzymes that create compatible ends.
What is star activity of Fermentas restriction enzyme?
*Star activity appears at 10-fold overdigestion (10 units x 1 hour). NR Buffer is not recommended, since star activity appears at 5-fold overdigestion (5 units x 1 hour). Restriction enzyme exhibits star activity under certain conditions. Denotes the possibility of thermal inactivation (at 65°C or 80°C in 20min).
How to select restriction enzymes for plasmid digestion?
Select restriction enzymes to digest your plasmid. *Pro-Tip* To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence analysis program such as Addgene’s Sequence Analyzer. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
Can a sequential Digest be performed with restriction endonuclease?
If there is no buffer in which the two enzymes exhibit > 50% activity, a sequential digest can be performed. Set up a reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer and incubate to completion.
How to optimize restriction enzyme digestion in Neb?
Should you require information on performing restriction enzyme digestions, please refer to Optimizing Restriction Endonuclease Reactions. You can also receive additional support by contacting [email protected]. We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free.