Transformation

Lab 4: Transformation and Plating Purpose Today you will perform heat shock transformation on chemically competent //E. coli //bacteria. Using a beaker of warm water you will force the bacteria to take in your ligated GFP-containing vector from the last lab. Materials Equipment Reagents •Ligation Reaction from last session • //E. coli //bacteria on ice • Tube of SOCS broth Methods The laboratory today will be the most time-consuming of all the lab sessions, so in order to finish on time you must be organized, quick, and careful. You will be using your ligation reaction from last week to transform //E. coli //bacteria, recover those bacteria, and then plate them on an agar plate containing arabinose. When handling live **//E. coli //bacteria, be sure not to touch the bacteria with any part of your body, and quickly dispose of any pipette tip, spreader, or transfer pipette that touches the bacteria in a trash can lined with a plastic bag. These //E. coli //bacteria do not cause disease, but you still should not get them on yourself. If your classroom has them, wear gloves and goggles when handling bacteria. ** 1. **//E. coli //bacteria should remain cold at all times, your instructor will remove the bacteria from the freezer and thaw them on ice. ** 2. Use your Air Displacement Pipette to 3. Let your bacteria sit on ice for 2-3 minutes to make sure they stay cold. 4.**Thaw your ligation reaction **from the last lab. 5. Use a pipette to **add 10.0μL of the ligation reaction to the bacteria. ** 6. Cap and flick the tube a couple times to mix and then put the mixture with bacteria on ice. 7. Leave the tube on **<span style="font-family: calibri,calibri;">ice for 20 minutes **<span style="font-family: calibri,calibri;">, optimally the tube would be left on ice for 1 hour. During this time **<span style="font-family: calibri,calibri;">re-watch the Chapter 4 Video **<span style="font-family: calibri,calibri;">. 8. While the bacteria DNA mixture is on ice, heat a beaker of water to 42<span style="font-family: calibri,calibri;">˚C or prepare your water bath 9. Incubate your bacteria for 60 seconds in the 42<span style="font-family: calibri,calibri;">˚ water. <span style="font-family: calibri,calibri;">Don’t let the bacteria spend much time at room temperature. Move them straight from the ice bucket into the water bath. 10. Remove the now-transformed bacteria from the water bath. Use the transfer pipette to add around 500μ of SOCS broth to the transformed bacteria. Place the bulb pipette on your bench to use again in ten minutes. Be sure not to mix your bulb pipette up with anyone else’s pipette. 11. Tightly cap the microcentrifuge tube containing your transformed bacteria. 12. Holding the transformed bacteria in your hand to warm them to 37<span style="font-family: calibri,calibri;">˚ C (body temperature) and shake the bacteria in an orbital motion for about a minute, then place the tube in the incubator at 37˚ C. You should come back <span style="font-family: calibri,calibri;">**<span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">tomorrow morning ** (at least 2 hours later) to plate the cells on the agar plate. 13. Write your name on the bottom of your agar plate in permanent marker. 14. Use the bulb pipette to add all of the bacteria in SOCS broth to the center of your agar plate and use the spreader to evenly distribute the liquid around the agar plate. Place the lid back on the plate. 15.<span style="font-family: calibri,calibri;">**<span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">Let the plate sit **for about a minute to absorb some of the liquid, then place it face up in the 37<span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">˚ C   incubator. 16. Tomorrow, after colonies have grown, the plates should to be moved from 37<span style="font-family: calibri,calibri;"><span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">˚ C to the refrigerator  to prevent overgrowth of bacteria. If you don’t see any colonies, try leaving the plates in the incubator for another day (also double check that the incubator is at the appropriate temperature). Clean-up Make sure that all of the tubes containing <span style="font-family: calibri,calibri;">//<span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">E. coli //bacteria are properly disposed of and that the ligation reaction is discarded. The microcentrifuge tube that contained your //<span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">E. coli // should be tightly capped before being thrown away. Be sure to put away the hot plate and beaker in addition to your pipette, as normal. Make sure the transfer pipette and spreader that you used are thrown away as well. Double check that your initials are on your agar plate and place it in the incubator. Hand in your group’s air displacement pipette to your instructor; you will not need it for the rest of the module. Conclusions Today you have inserted some circular plasmid DNA from a ligation reaction into <span style="font-family: calibri,calibri;">//<span style="font-family: Calibri,Calibri;"><span style="font-family: Calibri,Calibri;">E. coli // bacteria. By plating the bacteria on ampicillin containing plates, only the bacteria that have the ampicillin resistance gene from the plasmid DNA will be able to grow. By tomorrow you should be able to see individual colonies started by a single bacterium on the plate, and hopefully some of them will express GFP!
 * <span style="font-family: calibri,calibri;">2-20μL Air displacement pipette
 * • Pipette tips
 * • 1 transfer pipette
 * • 1 hot plate, beaker with water, and thermometer OR 1 water bath
 * • 1 tube float (optional)
 * • Ice bucket (One per lab table recommended)
 * • 1 microcentrifuge tube per student
 * <span style="font-family: calibri,calibri;">add 40.0μL of bacteria **<span style="font-family: calibri,calibri;">from the lab stock tube to your microcentrifuge tube

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