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, topAcysB phage DNA, and two samples of molecular weight markers (lambda DNA cut with HindIII, and lambda DNA cut with HindIII and EcoR1). The sizes of one of the molecular weight markers are shown on the picture below. There are also markers of 564 and 125 bp that are not shown in the picture.
The first step in the analysis is to prepare a standard curve to estimate the sizes of the DNA fragments. Measure the migration distance from the sample well to each of the bands in the molecular weight markers, and plot the size in base pairs as a function of migration distance on semi-log graph paper. Although not shown on the picture above, you also have a sample of lambda DNA digested with HindIII and EcoR1. The sizes of these markers are listed in a Table in Period 9B 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 the sizes of linear double-stranded DNAs.
These standards can also be used to estimaste the amount of DNA in a band. Given the table of sizes below, calculate the amount of DNA that is present in each band of the standards based on the total amount of DNA loaded on the gel.
Lambda/HindII sizes (bp) 23,130 9,416 6,557 4,361 2,322 2,027 564 125 Total 48,502 bp
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 the known amounts of DNA in each of the standards and comment on whether the correct amounts of DNA are present in Tube 1. You should also compare the mobilities of the DNA species in Tube 1 with the mobilities of the standard DNAs. What is the 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 controls?
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 species and calculate the size (by using your standard curve) and amount (by comparison to the 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 ther for restriction by one or both of the enzymes?
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 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? What is the size (bp) and form of the desired ligation product? Where would you expect to see it relative to the migration of the size standards?