BIO 467 MOLECULAR BIOLOGY OF PROCARYOTES LABORATORY Phosphatase Vector (if desired) Analyze Recovered DNA Set Up Ligation Reactions Suggested Time Frame: Week of October 21, 2002 GENERAL INFORMATION. Possibly the two most important types of enzymes for recombinant DNA work are the restriction enzymes and DNA ligases. We have talked about and used restriction enzymes, and you know what they do. Ligases catalyze the formation of a phosphodiester bond between two adjacent nucleotides. In nature ligases are important in DNA replication and in the repair of damaged DNA. In recombinant DNA work ligases are used to generate an intact molecule after annealing together the cohesive termini of two digested DNA fragments; without ligating, the phosphodiester backbone of the molecule will be "nicked" where the restriction fragments have annealed, and the cohesive termini can simply melt apart again leaving only restriction fragments rather than a new, intact DNA molecule. Remember, however, that not all restriction enzymes generate cohesive termini. Some enzymes generate blunt ended DNA; blunt ended DNA can also be ligated together, but this can be a more difficult proposition than ligating two fragments with sticky ends. Those of you who have selected SmaI may discover this! We will be attempting to ligate the fur DNA fragment into the pBluescript vector that we "opened up" with the double digestion. Having the fur fragment ligated into pBluescript will allow us to do a number of things; how many of these things can you think of? (Hint: Recall our discussion last week about the utility of the pBluescript vector!) Note that because our fur PCR product has unique restriction sites at each end, it can only be inserted into pBluescript in ONE ORIENTATION! Doing this type of ligation is often called “directional cloning”. Here’s something to think about: under what circumstances would you not have ligation in only one orientation? Prior to setting up the ligation reactions, however, you may want to treat the digested vector with an enzyme called a phosphatase. This enzyme will remove 5' phosphate groups from the digested vector. This is done to prevent the vector from ligating to itself ("recircularizing") without any insert DNA (our fur fragment), as the ligase cannot catalyze the formation of a phosphodiester bond without a 5' phosphate group. Why do we not want the vector to recircularize? Ligations can be a bit like PCR; many variables influence the success of ligation reactions, and even experienced molecular biologists can be frustrated by what often seems to be the unpredictable nature of ligations. One extremely important variable is the ratio between the vector DNA and the insert DNA. In the case of ligations involving fragments with cohesive termini, the vector and insert DNAs should be present in close to equimolar concentrations. For ligations involving fragments with blunt ends, the insert must be present in a molar excess. Because of these considerations, we will analyze our pBluescript and probe DNAs qualitatively and quantitatively prior to setting up the ligation reactions. We will talk more about how to set up the reactions in the class period. PROTOCOL 1. If you wish to phosphatase your vector you will need to start earlier. If you do not wish to phosphatase your vector, you may proceed directly to the gel analysis of your recovered DNA (Step 6). Dilute the phosphatase such that you can add about 0.01 Unit per picomole (pmol) of DNA "ends" containing 5' phosphate groups. A special “phosphatase dilution buffer” is used for this. If we assume you recovered all 2 ug of the pBluescript from the EcoRI digestion, can you determine how many pmols of "ends" you have? The answer should be "Yes!", but if you are not sure how to do this we will go over it in class. 2. To the tube containing your digested pBluescript add 6 ul of 10x phosphatase buffer, 3 ul water, and 1 ul of the diluted calf intestinal alkaline phosphatase (CIAP). Incubate at 37° C for 30 min. 3. While the phosphatase reaction is incubating prepare agarose gel(s). This time the gel(s) will be 1.0% agarose in TBE. 4. We don't want any CIAP to be carried over into the ligation reactions!! Do you know why? To ensure that no active CIAP winds up in the ligations, incubate the tube at 75° C for 15 min; this will heat inactivate the CIAP (what is the biochemical basis for this?) and ensure that our ligations have at least a chance to work! 5. While heat inactivating the phosphatase, you can prepare your fur DNA for the gel. Take 10.0 ul of your fragment, put into a microcentrifuge tube, and add 2.5 ul 5 x DNA Sample Buffer. 6. After heat inactivating the phosphatase, briefly spin that tube in a microcentrifuge and place on ice. Remove 10.0 ul to a new microcentrifuge tube and add 2.5 ul 5 x DNA Sample Buffer. 7. Run all the samples on the gel along with molecular weight markers. Run at 90 volts for about 1 hour. 8. Stain, destain, and visualize the gel. 9. Set up ligation reactions. You should set up several controls, both negative and positive! Can you determine what these controls will be? Try to think about how we could run a control for ligase activity, and control(s) to ensure that we had little or no vector recircularization (and no circular plasmid in your prep to begin with). The total volume of each reaction should be 10 ul; you will use a 5x Ligase Buffer (how many ul?), and 1 ul of the T4 DNA ligase itself (at least in those reactions where the ligase will be added). The remaining volume will be made up of the vector DNA, insert DNA, and, if necessary, water. We will talk about how much of the DNAs to add to obtain something close to an equimolar ratio of the two. The DNA, ligase buffer, and water should be added first; heat the tube at 42-45° C for 5 min, then plunge into ice and add the ligase. 10. If the fragments have at least one cohesive end, incubate overnight at 12-16° C. If both termini are blunt, incubate overnight at room temperature. [As mentioned earlier, the ratio of vector to insert DNA is crucial in ligation reactions. Having these amounts of both DNAs will maximize the chances that only one insert fragment will be ligated per vector molecule. Straying from this ratio can result in having linear arrays of the insert ("concatemers") ligated into the vector. For our purposes this would not be disastrous, but there are occasions when it is crucial to have only one insert molecule ligated into the construct. The reactions are run at 12-16° to promote annealing of the cohesive termini, which occurs more efficiently at these temperatures than at room temperature or higher; the ligase is still fairly active at these relatively low temperatures.] THE NEXT DAY On Thursday morning the instructor will put the ligation reactions at 4° until next week, when we will introduce our ligated products into E. coli cells through a process called transformation.