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One means of locating a region within a piece of DNA of unknown sequence is to
use restriction enzyme sites within the region as "signposts". By using a pair
of appropriate restriction enzymes, the relative position of all the
restriction sites within the target sequence can be mapped. The following
method will allow students to model the expected results for the exercises that
can be carried out in a school laboratory using the SAPS/NCBE kits (NCBE lambda
Kit and Plant DNA investigation kit); these kits allow students to investigate
the effects of EcoRI, BamHI and HindIII on pure
Lambda bacteriophage DNA (48502 bp long).
Load expt014. It contains 1 sample tube which contains Lambda DNA. Lambda is a bacterial virus. Its genome is well known.
Digest Lambda with EcoRI
Digest Lambda with BamHI
Double Digest of Lambda
You should now have four sample tubes: uncut, EcoRI digest, BamHI digest, and EcoRI and BamHI digests. Remember that the content of any tube can be checked by right clicking on the tube. The tubes can also be labelled using the same menu.
Analysis: Agarose gel electrophoresis
Before you can prepare the gel, you need to empty all tubes in the Reaction Tubes rack (by dragging them toward the waste bin).
The appearance of the gel shows what should be seen theoretically on a real gel - but remember that the precise conditions of running it will cause differences in the position of various bands (e.g. running time, percentage agarose, size of gel, voltage used). The effects of some of these factors can be investigated using the Agarose Gel Electrophoresis module.
Change the percentage of agarose to 0.5% to have a better separation of the bands
To simulate the difference of density of the bands with the size of the bands, click on a well, (top of track) and select intensity α.
Results and discussion
You should obtain a gel that looks like figure 1. To analyse the sizes of the bands, you can either use a size marker (there are plenty in the program) or click on a track to reveal the exact sizes of the bands.
You can have a go at working out the relationship between the restriction sites in the lambda genome.
Experiment written with Roger Delpech, SAPS.