Since Nanodrop concentration results for PCR and samples extracted from gels have proved unreliable, we have arrived at a sort of a default protocol for DNA of an uncertain concentration.

For a ligation or when large quantities of DNA are needed:

• DNA 15 microliters
• Restriction enzyme 1 @ 100x 1 microliters
• Restriction enzyme 2 @ 100x 1 microliters
• BSA (if needed) @ 10 x 5 microliters
• Demineralized water 28 microliters

For checking the presence of a restriction site on a gel:

• DNA 5 microliters
• Restriction enzyme 1 @ 100x 1 microliters
• Restriction enzyme 2 @ 100x 1 microliters
• BSA (if needed) @ 10x 5 microliters
• Demineralized water 38 microliters

The total volume should be 50 microliters

Incubation should ideally be at 37°C. When in doubt on the quantity of DNA used, favor a longer (20 to 30 minutes longer) incubation time than what is indicated on the NEB double digest guide. Avoid enzyme pairs with different incubation temperatures. This doubles the incubation time.

After the incubation period, the enzymes need to be heat-inactivated. Heat inactivation temperatures are usually on the order of 80 C. 20 minutes usually does the job.

The samples can then be frozen if needed.