Expt005 : Restriction Enzyme Digest

The aim of this experiment is to digest linear DNA fragments with a restriction enzyme

Method

Load Expt005. It contains four samples of DNA.

Check what the sample tubes contain.

• Load Sample 1 into a reaction tube (tube 1) and start the Agarose Gel Electrophoresis module. Load the sample onto the gel.

The gel shows that sample 1 contains a single fragment of DNA

• Close the Agarose Gel Electrophoresis module

Digest Sample 1 DNA .

• Click on Restriction Enzyme Digest (in the toolkit). This opens a new module we haven't used before.
• The new window contains a list box labelled "Select enzyme" containing a list of enzymes, a wide white rectangle in which the restriction site for the selected enzyme will be displayed, and a "Check List".

• Choose "EcoRI" from the list of available enzymes. When you choose an enzyme the restriction site that is recognised by that enzyme is displayed.
• Click successively the four check-boxes in the "Check List". These are the important steps one must perform to set an enzyme reaction. When the last check box is checked (incubate at correct temperature for an hour), an animation shows how the enzyme cuts the DNA. Click on the Return to BioLab button when finished.

As with every method in BioLab,all of the DNA in the reaction tubes has been processed, but none of the DNA in the sample tubes.

The reaction tube now contains reaction products: digested DNA.

Analyse digested DNA : Run a gel

It is useful to be able to see the undigested DNA next to the digested DNA on the same gel. The reaction tube rack now contains only one tube of digested DNA. Before you open the Agarose Gel Electrophoresis module, load some undigested DNA from sample 1 into one of the tubes in the reaction rack (e.g. reaction tube 2). Now, click on the Agarose Gel Electrophoresis method .

• Load the DNA from reaction tube 2 (undigested DNA) into track 1 of the gel.
• Load the DNA from reaction tube 1 (digested DNA) into track 2 of the gel.

Results and Discussion

EcoRI recognizes the DNA sequence GAATTC and cuts just after the G. After digestion with EcoRI, the reaction tube contains two fragments. These are the result of a single cut with EcoRI. Sample 1 has only one EcoRI site.

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