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DNA Strider Manual: Bio312 2000
Bonnie K. Baxter, Lewis & Clark College

In designing your analytical digests, you'll need to determine the size of the fragments that would be created by digesting your plasmid with a particular enzyme or pair of enzymes. You can, of course, do this by subtraction, since you know the size of the plasmid and the exact location of each site. The program can also do this for you if you like.

First, select the main sequence window to make it active. The choose "enzyme chooser" from the enz menu. Choose the enzymes you'd like to use by clicking on their names. If you'd like more than one, hold down "shift" as you click.

When you've finished, choose "digestion" from the enz menu. You'll get a window that shows you the enzymes you chose, the number of fragments created, and a list of those fragments in order of decreasing size, identifying the location and the sites at the end of each one.

How do we decide which enzymes to use?

Choose a single enzyme if possible, or at most a pair of enzymes. With five candidates to screen you can only do one digest per gel, so choose a digest that will give you as much information as possible.

You want a digest that can tell you whether your vector has an insert--the digest fragment pattern should be distinctly different in the vector alone vs. a recombinant plasmid.

You also want a digest that can tell you whether the insert you have is the one you expected. It should be the predicted size, and it should contain at least one predicted restriction site in the predicted position. Notice that you cannot tell the position of a site in a circular plasmid unless there is a fragment that contains that site at one end and a known site at the other, and that fragment is of a size that can be readily estimated from an agarose gel.

Finally, if you a planning a double digest, you'll want to choose a pair of enzymes that can work reasonably well together in the same buffer. Your Lab Manual appendix ("Background Information on Restriction Enzymes") lists the buffer requirements of each enzyme.

Why are there so few restriction enzymes on these maps?

For simplification, I have altered the restriction enzyme library file that DNA Strider uses to recognize restriction enzyme sites, so that it only recognizes the sites of 19 enzymes, all of which we have on hand. The original RE library file is also present in the DNA Strider folder--it's titled "Orig. RELibrary." If the name of this file were to be changed to "RELibrary" and Strider were rebooted, the entire library would appear.

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