Simply Cloning - Chapter 7 - Transformation скачать в хорошем качестве

Simply Cloning - Chapter 7 - Transformation

Simply Cloning is a video manual for making DNA constructs. Chapter 7 describes how to transform ligated DNA fragments into chemically competent E. coli cells. Narration Script: During the transformation step we are going to add the ligated plasmid to chemically competent E.coli cells, and expose the cell suspension to a heat shock. The heat shock will introduce the DNA inside the cells. Transformation protocol: • Thaw chemically competent cells on ice for 15 min • Add ligation mix to the competent cells, mix by tapping gently on the tube • Leave the cells on ice for 30 minutes • Heat-shock cells at 42 oC for 60 seconds • Put cells back on ice for 5 minutes • Add 4 volumes (800 µl) of LB medium without selective antibiotic • Shake for 45 min at 37 oC to develop antibiotic resistance • Spin down at 13000 rmp for 1 min • Remove 800 µl of supernatant, resuspend pelleted cells in the remaining LB • Plate on LB agar plate with antibiotic • I start the transformation by taking the competent cells out of a -80 oC freezer and thawing them on ice for 15 minutes. Each of these tubes contains 200 µl of competent cells prepared for one transformation. Notice that competent cells are sensitive to room temperature exposure and therefore I put them on ice the very moment I remove them from the freezer. After incubating the competent cells for 15 minutes on ice I am going to label the tubes as Vector plus Insert and Vector Control and I am going to add the corresponding ligation reaction to each of the tubes. Then I mix the DNA with cell suspension by gently tapping on the tube. I put the cells back on ice and leave them there for another 30 minutes. For the heat shock I transfer the tubes into a floating rack and put them into 42 oC water bath for 1 min. After the heat shock the cells go back on ice for 5 minutes. Now I am going to add 800 µl of LB medium without antibiotic. And I will shake the tubes in a 37 oC shaker for 45 minutes so the cells develop antibiotic resistance. Before plating I am going to reduce the volume back to 200 µl by spinning down the tubes in a microcentrifuge for 1 minute. Then I remove 800 µl of supernatant and resuspend the cells in the remaining 200 µl of LB. Now we are ready to plate the cells. For plating we will need a gas burner, two LB plates, a beaker with ethanol and a spreader. First, I am going to transfer the cells from the tubes to the plates. Then, I will burn the spreader, chill it down against LB agar without touching the cells and spread the cells around. Then I burn the spreader in flame, dip it in the ethanol, burn again, chill it against the plate, and spread the cells around. I will incubate the plates in the lid up position for 30 minutes at 37 oC to dry the liquid on the plate surface. Then I will flip them upside down and leave until the next morning. On the Day Two the first thing in the morning I rush to my plates to check for colonies. As you can see, I have about 30 colonies onthe Vector plus Insert plate and nothing on the Control plate, so I am pretty confident that most colonies contain the construct I want to make.