Ligation

Lab 3: Ligation Purpose Today you will ligate your amplified DNA from the previous lab into a linearized vector that you placed into an aliquot on the first day. This procedure should generate circular plasmids containing GFP DNA. Materials Equipment Reagents 1. **Label ** your Microcentrifuge tube with your initials using a permanent marker. 2. Take out the 5x Ligase Buffer to **thaw ** 3.**Add the Ligation Reagents to the tube ** a. Use a new pipette tip for each different reagent you use, discarding the old one in the trash. b. Add 4.0μL of your PCR reaction to the bottom of the new 0.2mL tube c. Add 6.0μL of Water to the tube d. Add 4.0μL of Linearized Vector to your mix e. Add 4.0μL of 5x Ligase buffer f. Go to your instructor’s lab table and add 2.0μL of DNA Ligase, which should be sitting on ice. g. Shake the tube gently so that all the components are mixed well 4.**Let the reaction sit overnight ** at room temperature 5.**Watch **the Chapter 4 video to get an idea of the techniques you will be using next lab. 6. The next morning, you or your instructor will put your reaction into the Thermal Cycler 7. Place your tube back in the plastic bag for your group after running the "Lig End" program. Clean-up After the reaction has been allowed to incubate overnight, be sure to put your ligation reaction in your group’s bag and place the bag back in the freezer. Extra 5x Ligase buffer, Linearized Vector, and PCR product can either be stored for another experiment or thrown away.
 * 2-20μL Air displacement pipette
 * • Pipette Tips
 * • 0.2mL (PCR) Tube
 * Amplified GFP PCR product (from last lab)
 * • Reagents from your group’s bag Linearized Vector
 * 5x Ligase Buffer
 * • DNA Ligase Enzyme
 * and run the "Lig End" program **, which heats up your sample to denature your enzyme, ending the reaction and improving the efficiency of transformation.
 * Store the ligation reaction **in the freezer until next lab period.

Conclusions Some of the pieces of linear DNA that you have mixed together in your reaction tube should have been joined together by DNA ligase into a circular plasmid. These circular pieces will remain mixed in with the un-ligated fragments until next lab session, when you will be putting the DNA into bacteria, and then only allowing the bacteria which have taken up the circular plasmid will grow.

Water || 4.0 6.0 || []
 * Methods
 * Reagent ** || Quantity (μL) ||
 * PCR Reaction
 * Linearized Vector || 4.0 ||
 * 5x Ligase Buffer || 4.0 ||
 * DNA Ligase || 2.0 ||
 * Total || 20.0 ||