The concentrations of RE and plasmid DNA need to be matched in a restriction digest. Manufacturers assay the activity of each batch of RE and express the activity in units of enzyme activity per μL. A unit of activity (U) is assessed in a standardized assay for measuring RE concentrations. Restriction digests are usually set up to contain at least 2-5 U per μg plasmid DNA. The ZyppyTM kits typically yields plasmid concentrations ranging from 10 to 30 ng/μL. (You will be able to estimate your plasmid DNA concentrations when you run the agarose gels in the next lab.) In this lab, we are using 7 μL of plasmid miniprep DNA in each reaction and 1 U of RE. This should be more than enough RE to ensure complete digestion of the plasmid DNA.
In your lab notebook, note which RE(s) you have decided to use.
Prepare a separate tube for each of your RE digests.
Combine the following components in each tube in order listed:
7.0 μL plasmid
1.0 μL 10X CutSmartTM buffer or 10X Buffer 3.1 (for HincII digests only)
2.0 μL (1.0 U) restriction enzyme
The total reaction volume should be 10 μL.
Ensure that the components of each reaction are well-mixed at the bottom of the tube by centrifuging them for a few seconds in the microcentrifuge.
Incubate the samples at 37 °C for at least 2 hr.
Store the reactions in the freezer.
Exercise 3 will be performed in the next laboratory session.