successful double digest

1. What is a double restriction digest?
2. What is single digestion and double digestion?
3. How long should a restriction digest take?
4. Why are there two bands in the digested sample?
5. Why is my restriction digest not working?
6. How can we prevent restriction digestion?
7. Are DNA fragments positive or negative?
8. What is a double digest electrophoresis?
9. What does it mean to digest DNA?
10. What happens if you add too much restriction enzyme?
11. Can I store a restriction digest?
12. How do you calculate restriction digest?

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 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.

Why are there two bands in the digested sample?

The two additional bands are probably not uncut plasmid forms (I run uncut plasmid DNA alongside) and are smaler then the expected band. Conditions, components and its concentration in the second reaction was the same as in the first.

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 can we prevent restriction digestion?

If further manipulations of the digested DNA are required, heat inactivation (raising the temperature to 65 or 80°C for 20 minutes) is the simplest method of stopping a reaction.

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 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 does it mean to digest DNA?

Experiment 1: Restriction Digestion

Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases (sometimes just called Restriction Enzymes or RE's).

What happens if you add too much restriction enzyme?

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.

Can I store a restriction digest?

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.

How do you calculate restriction digest?

Calculate the amount of each that you need to add to a restriction digestion in order digest 5ug (5000ng) of DNA with 5 units of enzyme. For example if my DNA is at 190 ng/ul, I would need: 5000ng/190ng/ul = 26 ul of my sample.