Why is my restriction digest not working?

Since a restriction enzyme may lose activity due to improper storage or handling, it is important that you always check the expiration date, verify that the enzyme has been stored at -20°C, and check the temperature of your freezer (do not allow temperatures to exceed -20°C, as multiple freeze-thaw cycles (more than 3 …

How do you store restriction enzyme 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.

Why should the restriction enzymes be placed in the freezer when not in use?

1) Keep restriction enzymes cold Restriction enzymes are proteins and are very sensitive to heat, and freezing and thawing.

What affects restriction enzyme digestion?

The digestion activity of restriction enzymes depends on the following factors: Temperature: Most endonucleases digest the target DNA at 37 °C with few exceptions. Some work at lower temperatures (~25 °C, Sma 1) while Taq I works at 65 °C.

How do you troubleshoot restriction enzyme based cloning problems?

Troubleshooting for Restriction Enzyme Based Cloning

  1. Check if your restriction enzyme is functional.
  2. Add extra nucleotides on the 5′ side of the restriction sites in your primers, and check the sequence of the restriction site to make sure there is no mutation.

How do you know if your restriction digestion was successful?

If the digested product would be visible at a lower coordinate on the gel, it would have made things easy. You can amplify your digested fragment with primer beginning in the flankers region and with only 3-4 bp in the intern 8680 bp region. If you do not get PCR fradments, was the digestion successfully.

Are restriction enzymes kept on ice?

Because enzymes are proteins and proteins denature as the temperature is increased, RE’s are always stored in a freezer until they are used. In fact, all of the ingredients in a Restriction Digest are kept on ice until it’s time for the reaction to begin.

Can you leave a restriction digest overnight?

Time-Saver qualified enzymes can cut substrate DNA in 5-15 minutes and safely digest overnight. For enzymes that are not Time-Saver Qualified, the recommended incubation time is 1 hr. In general, long incubations (several hours to overnight) are not recommended, unless digesting some gDNAs.

Can you store restriction enzymes at?

Storage at -20°C is recommended for most restriction enzymes. For a few enzymes, storage at -70°C is recommended for periods longer than 30 days.

How do you test for restriction enzymes?

Standard Protocol for Restriction Enzyme Digests

  1. Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence.
  2. Use the recommended buffer supplied with the restriction enzyme.
  3. Clean up the DNA to remove any contaminants that may inhibit the enzyme.

What happens if you use too much restriction enzyme?

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.

Why would a restriction enzyme not cut?

There can be several different reasons why your restriction enzyme does not cut the DNA as reviewed in this video. The preparation of DNA to be cleaved should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction enzyme activity.