EXERCISE 9B Name How are plasmids used in recombinant DNA technology Day Two Which unknown plasmid sample had no plasmids which had non recombinant pUC18 plasmids and which had recombinant pUC18 plasmids Objectives After completing this exercise you should be able to Analyze an agarose gel to determine the number of different sized DNA fragments present in a sample Analyze an agarose gel to determine the approximate size of each DNA fragment Determine which agar plates were inoculated with transformed bacteria and which were inoculated with bacteria that were not transformed Examine colonies of transformed E coli to identify the colonies that carry a non recombinant plasmid and the colonies that carry a recombinant plasmid Prelab Before you come to lab read this entire exercise The separation of DNA molecules by electrophoresis is similar to the separation of proteins by electrophoresis In both cases the molecules to be separated are loaded onto a support medium and then subjected to an electrical field The cathode negative electrode attracts positively charged molecules while the anode positive electrode attracts negatively charged molecules This attraction causes the molecules in the mixture to migrate through the support medium at different rates depending on their size shape chemical composition and electrical charge As the molecules migrate at different rates they gradually separate from each other The main difference between protein electrophoresis and DNA electrophoresis lies in the composition of the support medium While polyacrylamide gels are generally used for protein separations agarose gels are commonly used for DNA separations Agarose is a polysaccharide that is extracted from seaweed When agarose is dissolved in a solution at high temperature it has a liquid consistency but as the solution cools it turns into a gel Agarose gels are cast by dissolving agarose in a Tris buffered solvent at a high temperature and then pouring the solution into a horizontal tray and allowing it to cool A comb is inserted into the cooling gel so that wells are formed as the gel solidifies The cooled gel is then transferred into a gel box where it is submerged in a Tris buffered electrode solution before loading DNA samples into the wells Since these gels are run horizontally and beneath the electrode buffer they are sometimes referred to as horizontal gels or submarine gels DNA molecules will migrate towards the anode during electrophoresis because they have a high concentration of negatively charged phosphate groups in the sugar phosphate backbone of the double helix These negative charges provide a fairly uniform charge to mass ratio among all DNA molecules Because of this uniform ratio the migration rate of DNA molecules during agarose gel electrophoresis is almost entirely a function of size As with protein electrophoresis smaller molecules move through the gel more rapidly than larger molecules Scientists generally measure the size of DNA molecules in terms of number of base pairs bp rather than molecular weight MW Exercise 9B Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 9B 1 Recall that during SDS PAGE there is a linear relationship between the migration distance of the proteins and log of MW Similarly during agarose gel electrophoresis there is a linear relationship between the migration distance of the DNA molecules and log of bp number of base pairs Because of this agarose gel electrophoresis can be used to estimate the size number of base pairs of DNA molecules In order to do this a standard curve that shows the relationship between migration distance through the gel and log of bp must be generated The standard curve is generated by measuring the migration distance of several DNA molecules of known size number of base pairs plotting a scatter diagram of migration distance vs log of bp and then using linear regression to determine the equation of the best fit straight line for the data points Once this is done you can substitute the migration distance of any DNA molecule on your gel into the linear regression equation and then calculate the log of bp for that molecule DNA like most proteins is colorless Therefore before DNA samples are loaded onto an agarose gel they are mixed with a sample buffer that includes a blue tracking dye During electrophoresis the tracking dye migrates very rapidly through the gel along with the smallest of DNA fragments When the dye approaches the end of the gel you know it is time to stop the electrophoresis Glycerol is also included in the sample buffer in order to make the sample denser so that it settles into the well as it is loaded After electrophoresis is complete the gel is stained so that the DNA bands can be seen The most commonly used dye for staining DNA is ethidium bromide a dye that is highly specific for binding to DNA or RNA DNA or RNA bound to ethidium bromide fluoresces strongly under ultraviolet light so these gels are viewed and photographed using ultraviolet light Because both ethidium bromide and ultraviolet light are mutagens you will be using a less sensitive but safer stain for your DNA gels methylene blue Methylene blue is a blue colored stain that binds to DNA in the same way that Coomassie Blue binds to proteins The blue color is easily seen using normal visible light What is the goal of this lab exercise During exercise 9A you were given 3 unknown plasmid samples labeled A B and C These samples were used to carry out 2 experiments 1 Competent E coli cells were mixed with the 3 unknown samples in order to allow transformation to take place The E coli cells were then plated on nutrient agar containing ampicillin and Xgal 2 The restriction enzyme EcoRI was mixed with the 3 unknown samples in order to cut any DNA present into fragments The resulting DNA fragments were then separated using agarose gel electrophoresis In this lab exercise you will view the results of these 2 experiments in order to determine which unknown plasmid sample had no plasmids which sample had non recombinant pUC18 plasmids and which sample had recombinant pUC18 plasmids In addition you will use the results of agarose gel electrophoresis to estimate the size of the fragments that were produced when EcoRI digested the DNA in the 3 unknown samples Exercise 9B Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 9B 2 Lab Procedures I Examine your DNA gel in order to determine which unknown plasmid sample had no plasmids