Expt014: Restriction mapping of the Lambda bacteriophage genome

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).

More on Lambda

Method

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

Transfer some DNA from sample 1 into a Reaction Tube.

Start the Restriction Enzyme Digest module by clicking on the relevant icon in the Methods list.

Select EcoRI in the list of enzymes.

Perform the digest as usual by checking the relevant boxes in succession.

Return to BioLab.

Transfer some DNA from the Reaction Tube into a sample tube (Sample 2) to save the sample, and discard the contents of the Reaction Tube.

Digest Lambda with BamHI

Perform the same protocol but choose BamHI in the list of enzymes.

Save the reaction in a Sample Tube (Sample3)

Double Digest of Lambda

Transfer some DNA from sample 2 (Lambda digested with EcoRI) into Reaction Tube 1.

Use the Restriction Enzyme Digest module to digest this sample with BamHI.

Save the digested DNA into sample 4.

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).

Transfer some DNA from samples 1-4 into Reaction Tubes 1-4 respectively.

It is useful to movethe Reaction Tubes rack to the top of the lab bench.

Click the Agarose Gel Electrophoresis method.

To load the gel simply click on a Reaction Tube and drag it to the top of a track (the well). When you release the mouse button, the DNA is loaded into the gel.

Repeat the process for reactions 2-4 using lanes 2-4 respectively.

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.