restriction mapping double digest problem

1. Why are there double digestion restrictions?
2. Why is my restriction digest not working?
3. What happens if you add too much restriction enzyme?
4. Can two restriction enzymes cut at the same site?
5. How do you calculate restriction digest?
6. How long should a restriction digest take?
7. Is heat inactivation of restriction enzymes necessary?
8. Do restriction enzymes expire?
9. How can restriction digest be prevented?
10. Why would a restriction enzyme not cut?
11. How do you select restriction enzymes?
12. Why do restriction enzymes need to be kept on ice?

Why are there double digestion restrictions?

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.

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.

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 two restriction enzymes cut at the same site?

You can do with proper controls like single restriction digestion and both the enzymes together to make sure that both are cutting independently and together as well and go ahead with your plans. Unfortunately I was restricted to these two enzymes that happened to have sites next to each other!

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.

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.

Is heat inactivation of restriction enzymes necessary?

FAQ: Is it necessary to inactivate restriction enzymes after vector digestion? Inactivation of restriction endonucleases is generally not necessary, but in some cases it might increase the transformation efficiency.

Do restriction enzymes expire?

All enzymes are assayed for activity every 3-6 months; the expiration date is given on the label attached to each vial of enzyme. After thirty-five years of experience with restriction enzymes, we have found that most are very stable when stored at -20°C in the recommended storage buffer.

How can restriction digest be prevented?

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.

Why would a restriction enzyme not cut?

FAQ: Why is my Restriction Enzyme not cutting DNA? ... 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. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.

How do you select restriction enzymes?

When selecting restriction enzymes, you want to choose enzymes that:

1. Flank your insert, but do not cut within your insert.
2. Are in the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid.

Why do restriction enzymes need to be kept on ice?

Do enzymes really need to be kept on ice all of the time? ... Enzymes need to be stored and used at their optimum temperatures. Enzymes are generally stored in glycerol at -20C. This prevents them from freezing completely, which causes protein denaturation and results in a loss of activity.