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 BamHI restriction enzyme?
BamHI (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site. DNA is bound in a large cleft that is formed between dimers; the enzyme binds in a “crossover” manner.
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.
What is single digestion and double digestion?
Single-digested plasmids are digested with a single restriction enzyme, which results in compatible ends with self-ligation. On the other hand, double-digested plasmids are digested with two different restriction enzymes. Therefore, the two ends are not compatible for self-ligation.
What is the purpose of restriction enzyme digestion?
Restriction digestion is usually used to prepare a DNA fragment for subsequence molecular cloning, as the procedure allows fragments of DNA to be pieced together like building blocks via ligation.
What is the principle of restriction enzyme digestion?
Principle: Restriction Digestion involves fragmenting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases commonly known as Restriction Enzymes (RE). Because of this property the restriction enzymes are also known as molecular scissors.
Why is a buffer added to a restriction digest?
Buffers containing low concentrations of EDTA (1mM) are often used to protect DNA from nuclease degradation during storage, but EDTA can interfere with restriction enzyme digestion if the final concentration in the reaction is too high.
What do you understand by double digestion?
How do restriction enzyme digests work?
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.
What are the reaction conditions for BamHI restriction enzyme?
Thermo Scientific BamHI restriction enzyme recognizes G^GATCC 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 much BamHI is needed to digest 1 µg of DNA?
Unit Definition One unit is defined as the amount of BamHI required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl..
How long does it take to digest FastDigest BamHI?
Incubation at 37°C. 1 µL of FastDigest BamHI is formulated to digest up to: – 1 µg of lambda DNA in 5 min. – 1 µg of plasmid DNA in 5 min. – 0.2 µg of PCR product in 5 min. – 1 µg of genomic DNA in 5 min, or 5 µg of genomic DNA in 30 min. Thermal Inactivation: Incubation at 80°C for 5 min.
Can a Thermo Scientific buffer be used for double digestion?
For optimal results with fast reaction and 100% buffer compatibility, we highly recommend using FastDigest restriction enzymes in double digestion. In certain cases however, it may be possible to perform double digestion using a mix of Thermo Scientific conventional and Fastdigest restriction enzymes.