What is a double restriction digest?
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 you test for double digestion?
Digestion: 1ug of DNA+2uL of Cut smart buffer (10x if your using NEB product)+1uL of each enzyme (according to your mentioned stock conc.)+ rest make up with sterile milliQ. Incubate at 37oC for 3 hrs, estimate the digested products concentration. Verify that both enzymes using the same reaction buffer.
What is the purpose of a double digest?
A double digest is one where two restriction enzymes are used to digest DNA in a single reaction.
How do you do restriction digestion?
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.
What is the difference between single and double digest?
In single-digested plasmids, digestion with the same restriction enzyme produce both ends while, in double-digested plasmids, digestion with a different restriction enzyme produce each end. Hence, this is an important main difference between single digested plasmid and double digested plasmid.
How does restriction digest work?
How do restriction enzymes work? Like all enzymes, a restriction enzyme works by shape-to-shape matching. When it comes into contact with a DNA sequence with a shape that matches a part of the enzyme, called the recognition site, it wraps around the DNA and causes a break in both strands of the DNA molecule.
Can I freeze a restriction digest?
If you don’t have time to gel purify your reaction you can still put it in the freezer overnight. If the enzyme is still active I wouldn’t put it at 4C. You might want to heat-inactivate the enzyme to avoid star activity while you are freezing/thawing – generally by heating it to 65/70C for about 20 minutes.
What is the doubledigest calculator?
DoubleDigest Calculator—Thermo Scientific Easily determine optimal reaction conditions for your double digest reaction using this tool. DoubleDigest conveniently calculates the best enzymatic reaction buffer, enzyme concentrations, incubation conditions, and any additives needed in your double digest reaction.
How does XbaI work?
XbaI cleaves to leave a 5′ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI or NheI. Blocked by overlapping dam methylation.
What does XbaI mean in E coli?
An E. coli strain that carries the XbaI gene from Xanthomonas badrii (ATCC 11672). One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam – /HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 µl.
How do I set up a Double Digest in cutsmart buffer?
Setting up a Double Digestion. 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.