Wittrup: Restriction digestion

From OpenWetWare

Jump to: navigation, search

Materials

DNA

Restriction enzyme(s)

Compatible buffer

37˚C water bath

General Protocol

The specific protocol can vary depending on the particular restriction enzyme. The amount of enzyme needed (per μg of DNA) and the optimal temperature are generally available on the product specification sheet and/or the website. The New England Biolabs website ([1]) provides useful information on a variety of issues relating to DNA digestion.

1. Add ddH2O to tube (often 20-50 μL total volume).

2. Add restriction digest buffer to tube. Mix well (often provided as 10x concentrate).

3. Add BSA (bovine serum albumin) if necessary (some enzymes require BSA for optimal activity).

4. Add DNA to tube. Mix well.

5. Add restriction enzyme to tube. Mix well.

6. Incubate at appropriate temperature for required time.

7. Heat inactivate the enzyme if desired and if possible.

Specific Example

1. Add 16.6 μL of ddH2O to a microcentrifuge tube.

2. Add 2 μL of 10x NEBuffer 2. Mix well.

3. Add 0.2 μL of 100x BSA. Mix well.

4. Add 1 μL of DNA (0.5 μg/μL). Mix well.

5. Add 0.2 μL of NheI (10 U/μL). Mix well.

6. Incubate at 37ºC for 2 h.

7. Incubate at 65º for 20 min. to heat inactivate.

Personal tools