What is restriction enzyme double digestion?
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.
Why is double digestion carried out?
Time Saving. The recombinant fragments of single-digested plasmids have to be selected for their proper orientation while the double-digested plasmids ensure the proper orientation of the foreign DNA fragment. Therefore, double-digested plasmids save time in the recombinant DNA techniques, not single-digested plasmids.
Can you mix restriction enzymes?
Alternatively, you can productively digest with fewer units of enzyme for up to 16 hours with many restriction enzymes. An extremely important, yet often overlooked, element of a successful restriction digest is mixing. The reaction must be thoroughly mixed to achieve complete digestion.
How do you stop restriction digest?
At NEB, we use the following stop solution: 50% glycerol, 50 mM EDTA (pH 8.0), and 0.05% bromophenol blue (10 μl / 50 μl reaction). If further manipulations of the digested DNA are required, heat inactivation (raising the temperature to 65 or 80°C for 20 minutes) is the simplest method of stopping a reaction.
Can you run a restriction digest overnight?
Time-Saver qualified enzymes can cut substrate DNA in 5-15 minutes and safely digest overnight. For enzymes that are not Time-Saver Qualified, the recommended incubation time is 1 hr. In general, long incubations (several hours to overnight) are not recommended, unless digesting some gDNAs.
What is the purpose of digesting the PCR product with restriction enzymes?
This cloning will use a sticky end ligation strategy. First, the PCR product will be digested with restriction enzymes (BamHI & HindIII) to generate sticky ends; then ligated appropriately. The PCR reaction itself contains not only the PCR product (DNA) but also surviving DNA polymerase, and remaining nucleotides.
How do you do restriction digestion?
Restriction digestion is accomplished by incubation of the target DNA molecule with restriction enzymes – enzymes that recognize and bind specific DNA sequences and cleave at specific nucleotides either within the recognition sequence or outside of the recognition sequence.
How many restriction enzymes should be used?
In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.
How long do restriction enzymes take to work?
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 KpnI restriction enzymes?
Thermo Scientific KpnI restriction enzyme recognizes GGTAC^C sites and cuts best at 37°C in its own unique 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.
How do I select a reaction buffer for my Double Digests?
NEB’s online tools, NEBcloner and Double Digest Finder will help guide your reaction buffer selection when setting up double digests. 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.