double digestion protocol

1. What is a double restriction digest?
2. What is a double digest electrophoresis?
3. What is single digestion and double digestion?
4. How long should a restriction digest take?
5. What are the steps in restriction digestion?
6. Are DNA fragments positive or negative?
7. What enzyme digests DNA?
8. What is the purpose of gel electrophoresis?
9. What is star activity in restriction digestion?
10. What is a DNA restriction map?
11. Why is my restriction digest not working?
12. How do you store digested plasmids?

What is a double restriction digest?

Double Digests. Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. ... The Performance Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers.

What is a double digest electrophoresis?

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 single digestion and double digestion?

Single digest: one restriction enzyme only. Double digest: two restriction enzymes.

How long should a restriction digest take?

*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. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours.

What are the steps in restriction digestion?

Restriction Enzyme Digest Protocol

1. Add components to a clean tube in the order shown: ...
2. Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
3. Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.
4. The digested DNA is ready for use in research applications.

Are DNA fragments positive or negative?

DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.

What enzyme digests DNA?

ka. Restriction Endonucleases or Restrictase) is an enzyme that digests or cuts the DNA into pieces.

What is the purpose of gel electrophoresis?

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.

What is star activity in restriction digestion?

Star activity is the relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimal for the enzyme.

What is a DNA restriction map?

Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.

Why is my restriction digest not working?

Incomplete or no digestion due to enzyme activity blocked by DNA methylation. If your enzyme is active and digests the control DNA and the reaction is set up using optimal conditions, but you still see issues with digestion, it might be because the enzyme is inhibited by methylation of the template DNA.

How do you store digested plasmids?

The product of restriction digestion can be easily stored at -20 C. At 4 C it would be fine but to ensure that there is no activity and no star activity it is recommended to keep it at -20 C.