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.