Table of Contents
What is a double restriction digest?
A double digest is one where two restriction enzymes are used to digest DNA in a single reaction.
What is single digestion and double digestion?
Single-digested plasmid refers to a plasmid digested by a single restriction enzyme while double-digested plasmid refers to a plasmid digested by two different restriction enzymes.
What does double digest mean in biology?
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.
What is restriction digest used for?
Restriction enzyme digestion is commonly used in molecular cloning techniques, such as PCR or restriction cloning. It is also used to quickly check the identity of a plasmid by diagnostic digest.
Why do we use two restriction enzymes?
Using two different restriction enzyme sites can help ensure the correct orientation of the gene of interest when it is inserted and prevent the plasmid vector from ligating with itself.
Why do we do double digest?
So, if the two RE’s are compatible bufferwise you might do a double digest in one go, but depending on the compatibility of your two RE’s of choice, you might need to do it in two phases: stop the reaction of the first digest (heat-shock), purify the DNA-digest, then do the second digest…
Why do we digest PCR products?
For cloning applications, purification of PCR products prior to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the yield of ligation.
Why do we do double digestion?
Can I digest PCR product directly?
Frequently, a PCR product must be further manipulated by cleavage with restriction enzymes. For convenience, restriction enzyme digestion can be performed directly in the PCR mix without any purification of the DNA.
What is digestion after PCR?
The most convenient option for digestion of PCR-ampli- fied DNA is the addition of a restriction enzyme directly to the reaction tube after completion of PCR. The majority of restriction enzymes are active in PCR buffers. However, digestion of PCR products in the amplification mixture is often inefficient.