how much should i using dna restriction enzymes treatment

In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest. NEB has introduced a line of High-Fidelity (HF®) enzymes that provide added flexibility to reaction setup. Some restriction enzymes require more than one recognition site to cleave efficiently.

Full Answer

How much DNA is needed to make a restriction enzyme?

A "Typical" Restriction Digest Restriction Enzyme 10 units is sufficient, generally 1 µl i ... DNA 1 µg 10X NEBuffer 5 µl (1X) Total Reaction Volume 50 µl Incubation Time 1 hour* 1 more rows ...

Where can I find a list of restriction enzymes?

For a list of many commonly used restriction enzymes, visit NEB. 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.

How much overdigestion of DNA is required for restriction endonuclease reactions?

However, most researchers follow the "typical" reaction conditions listed, where a 5–10 fold overdigestion is recommended to overcome variability in DNA source, quantity and purity. NEB offers the following tips to help you to achieve maximal success in your restriction endonuclease reactions.

How fast do restriction enzymes break down substrates?

By conventional definition, one unit of restriction enzyme cleaves 1 μg of a defined substrate (e.g., plasmid pUC19) to completion in 1 hour in 50 μL under optimal conditions.

What is the purpose of a control DNA?

Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability. If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample.

What is the amount of enzyme volume needed to prevent star activity?

Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol. Additives in the restriction enzyme storage buffer (e.g., glycerol, salt) as well as contaminants found in the substrate solution (e.g., salt, EDTA, or alcohol) can be problematic in smaller reaction volumes.

How long can a 10x ne buffer be stored?

Please refer to the enzyme's product page for storage information. 10X NEBuffers should also be stored at -20°C.

Does DNA methylation inhibit digestion?

Extra wash steps during purification are recommended. Methylation of DNA can inhibit digestion with certain enzymes. For more information about methylation, visit Effect of CpG Methylation on Restriction Enzyme Cleavage and Dam and Dcm Methylases of E.coli.

What is CpNpG methylation?

In mammalian and plant systems, CpG or CpNpG methylation is a common DNA modification with implications in biological processes, making it a major focus of epigenetic studies. Sensitivity of restriction enzymes towards methylated DNA recognition sites depends on the restriction enzymes.

What happens when restriction enzymes are binding to DNA?

Restriction enzyme binding to the DNA may result in bands or smears above the expected bands in electrophoresis. Both star activity and incomplete digestion also give rise to unexpected patterns on the gel. Therefore, it is essential to differentiate the two for troubleshooting.

How long does it take for a restriction enzyme to digest?

This is especially crucial when “fast” restriction enzymes, which are designed to complete a digestion in 5–15 minutes, are used in place of “conventional” enzymes with 1-hour incubation times.

What is incomplete digestion?

Incomplete digestion. Incomplete digestion is a frequently encountered issue when using restriction endonucleases. Incomplete digestion may occur when too much or too little enzyme is used. The presence of contaminants in the DNA sample can inhibit the enzymes, also resulting in incomplete digestion.

What are the parameters of restriction enzyme reactions?

Important parameters to consider include substrate (DNA) and enzyme amounts, reaction volume, and incubation time.

What enzymes bind to DNA?

Some restriction enzymes (e.g., FokI, TauI) may bind tightly to the DNA, resulting in an apparent gel shift in electrophoresis. Using a loading dye containing SDS, and heating to dissociate the enzymes from the cleaved DNA, can prevent such gel shifts (Figure 2). Figure 2.

What is the function of DNA methyltransferases?

DNA methyltransferases drive the methylation reaction by transferring a methyl group from a donor to the acceptor bases (e.g., A and C). The most common types of DNA methyltransferases found in laboratory strains of bacteria include:

What is restriction enzyme digestion?

Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. For a list of many commonly used restriction enzymes, visit NEB .

How long to digest DNA for cloning?

For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours. *Pro-Tip* If you will be using the digested DNA for another application (such as a digestion with another enzyme in a different buffer), but will not be gel purifying it, ...

What enzyme to use to remove overhang?

Use T4 DNA Polymerase or Klenow DNA Polymerase I for 3′ overhang removal and 5′ overhang fill-in. If you are using blunt ends or a single enzyme to cut the vector, you will need to use a phosphatase to prevent re-circularization of the vector if you are cloning in an insert.

How long to incubate DNA?

*Pro-Tip* Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient.

Can a plasmid be resistant to cleavage?

Plasmids grown in Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites. See NEB's table of methylation sensitive restriction sites. Sometimes enzymes cut sequences which are similar, but not identical, to their recognition sites.

How much of the DNA solution volume should be added to the reaction for enzymes?

To prevent this, the DNA solution volume added to the reaction for these enzymes should be no more than 25% of total reaction volume. Cleanup of the PCR fragment (e.g., Monarch ® PCR & DNA Cleanup Kit) prior to restriction digestion is recommended. PCR components can inhibit enzyme activity.

What enzymes are salt sensitive?

Enzymes that have low activity in salt-containing buffers ( NEBuffer 3.1 or NEBuffer r3.1) may be salt-sensitive. DNA purification procedures that use spin columns can result in high salt levels, which can inhibit enzyme activity. To prevent this, the DNA solution volume added to the reaction for these enzymes should be no more than 25% ...

How many sites are needed for enzymes to digest?

Some enzymes require two sites for optimal digestion. When a single recognition site is present in a sequence, the second site can be added to the reaction in the form of a short double-stranded oligo containing one, or several, copies of the enzyme’s recognition site.

Does PCR inhibit enzymes?

PCR components can inhibit enzyme activity. In addition, the polymerase present in the PCR is active during the digestion step, and can modify the newly created ends by blunting them. Some enzymes may bind tightly to the substrate DNA.

Enzyme

DNA

Reaction Volume

Incubation Time

Stopping A Reaction

Storage

Stability

Control Reactions