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Learning Module - Experiment 5B

Electrophoretic Analysis of Restricted DNAs and Products of Ligation

In this lab, the success of the subcloning performed in Experiment 4B will be analyzed by agarose gel electrophoresis.

Remember that during the subcloning experiment, samples from various stages of the procedure were set aside for later analysis by gel electrophoresis. These samples will be electrophoresed along with standards of pTrc99A, λZAP/topAcysB bacteriophage DNA, and two samples of size markers (λDNA digested with HindIII, and λDNA digested with HindIII and EcoR1). The sizes of the standard markers are given in the Coursepack.

Gel showing band migration.

Be sure to label your gel completely and provide a Legend (do not make any marks on the gel picture itself)

Prepare a standard curve for linear double-stranded DNA

The first step in the analysis is to prepare a standard curve to estimate the sizes of3the DNA fragments in your samples. Measure the migration distance from the sample well to each of the bands in the size markers, and plot the size in base pairs (log scale) as a function of migration distance (linear scale) on semi-log graph paper. The sizes of these markers are listed in a table in Experiment 5B of the Laboratory Manual. Draw a smooth curve through the points and use this graph to estimate the sizes of the DNAs in the other samples. Recall that the size standards are linear double-stranded DNA, and can only be used to estimate accurately the sizes of linear double-stranded DNAs.

These markers can also be used to estimate the amount of linear double-stranded DNA in a sample band. Given the sizes shown on the table in the Coursepack and the total amount of DNA in each standard loaded on the gel, calculate the amount of DNA that is present in each band of the size markers.

Compare your sample from "Tube 1" to the controls in Lanes 1 and 2

Calculate the amount of each DNA that should be in Tube 1 based on the amount that was in the sample prepared for restriction endonuclease digestion and the volume that was set aside in Tube 1. Compare your sample to the known amounts of DNA in each of the control DNAs (Lanes 1 and 2) and comment on whether the correct amounts of DNA are present in your Tube 1. You should also compare the mobilities of the DNA species in Tube 1 with the mobilities of the control DNAs. What is the topological form of each DNA from Tube 1 (i.e. single- or double-stranded, relaxed, supercoiled, linear) and are these what is expected when compared to the known topological forms in the controls

Analyze the sample from "Tube 2"

Tube 2 contains the sample that was set aside after digestion with SacI and XbaI. What DNA species are expected to be present after digestion? Identify each of the observed bands and calculate the size (by using your standard curve) and amount (by comparison to the size standards) of each DNA species. Are they consistent with what is expected based on the volume of the digestion reaction that you used from Tube 2 for electrophoresis? What, if any, evidence is there for restriction by one or both of the enzymes?

Analyze the sample from the ligation ("Lig")

Is there evidence of ligation? How do you know? How many different ligation products would be expected if each of the starting DNAs were cut by one or both of the restriction enzymes? With this in mind, do you expect to see enough of any one of these ligation products to be visible on the gel as a distinct band? If distinct bands are present after ligation, speculate on their identity. What is the size (bp) and topological form of the desired ligation product? Where would you expect to see it on the gel relative to the migration of the control DNAs and double-stranded size standards?