Quick and dirty digestion protocol for DNA that has concentrations on the order of 80ng - 30ng / microliter

Since nanodrop concentration results for pcr and samples extracted from gels have proved unreliable we have arrived at a sort of default protocol for DNA of an uncertain concentration. From the ligation reaction setup and success rates we have deduced concentrations for pcr products or plasmid backbones extracted from gels are in the 30 - 80 ng / microliter range and this protocol seems to work fine.

For a ligation or when large quantities of digested DNA are needed.

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

For checking restriction site presence 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 50ⷧⷧ microliters

Incubation should ideally be at 37 C. When in doubt on the quantity of DNA used, favor a longer incubation time. 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 70 C. 10 minutes usually does the job. Check for the specific enzymes used on the [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp#.UCKBwHiuJ2M neb website]

The samples can then be frozen if needed.