Digest

1. Mix 5 to 10µg of DNA, 4µL of restriction enzyme, 4.5µL of appropriate buffer in a PCR tube. Add sterile water to bring up to 20µL total.

2. Incubate at optimal temperature for ~2 hours.

NotI incubates at 37^oC. BsmBI incubates at 55^oC. You can search enzymes in the plasmid digest to see their incubation temperatures.

Gel

3. Pour 1% agarose into a gel form. Place an appropriate well comb into the gel form.

4. Add 2.5µL of Ethidium Bromide to the liquid agarose gel. Move any bubbles to the side of the gel with a pipette tip.

5. Add 1µL of 6x purple loading dye for every 5µL of digest mix to the PCR tubes.

For a 20µL mix as above, add 4µL of purple loading dye.

6. Add 8µL of DNA ladder to one of the wells.

For this entire protocol, make sure you know what you put in each well.

7. Add all of each digest to each well.

e.g. add 12µL of digest to one well, 12µL of the next digest to the next well, etc.

8. Extract and gel purify the appropriate DNA bands.

Benchling's Virtual Digest tool is a great help for this.