Lesson 2: Transformation Laboratory

Transformation Procedure  (Christina)

1. Two closed micro test tubes were obtained, one was labeled +pGLO and the other labeled –PGLO. Both test tubes were labeled with your groups names and placed in a foam tube rack.
2.  Both test tubes were opened and using a sterile transfer pipet, 250 µl of transformation solution (CaCl₂) was transferred into each test tube.
3. The test tubes were placed on ice.
4. A sterile loop was used to pick up 1-4 large colonies of bacteria from your starter plate. Starter colonies that were “fat” (ie. 1-2mm in diameter) were selected. It was important to take individual colonies and not just a swab of bacteria from the dense portion of the plate, considering the bacteria must be actively grown to achieve high transformation efficiency. Only uniformly circular bacteria with smooth edges were chosen. The +pGLO tube was picked up and the loop was immersed into the transformation solution at the bottom of the tube. The loop was spun between your index finger and thumb until the entire colony was dispersed in the transformation solution (no chunks floated). The tube was placed back into the tube rack in the ice. This was repeated for the –pGLO using a new sterile loop.
5. The pGLO DNA solution with the UV lamp was examined. Observations were noted. A new sterile loop was immersed into the pGLO plasmid DNA stock tube. A loopful was withdrawn. There was a film of plasmid solution across the ring. Similar to seeing a soapy film across a ring for blowing soap bubbles. The loopful was mixed into the cell suspension of the +pGLO tube. A pipet of 10 µl of pGLO plasmid was mixed into the +pGLO tube, optionally. DNA plasmid was not added to the –pGLO tube. Both the +pGLO and –pGLO tubes were closed and returned back to the ice rack.
6. The tubes were incubated on ice for 10 minutes. The tubes were pushed all the way down in the ice rack so the bottom of the tubes stick out and made contact with the ice.
7. Your LB nutrient ager plates were labeled on the bottom (not the lid) as follows: LB/amp plate labeled: +pGLO, LB/amp/ara plate labeled: +pGLO, LB/amp plate labeled:  -pGLO, LB plate labeled: -pGLO.
8. Heat shock. Both the (+) pGLO  and (-) pGLO tubes were transferred from the foam rack into the water back, that was set at 42°C, for exactly 50 seconds. The tubes were pushed all the way down in the rack so the bottom of the tubes stick out and are in contact with the warm water. The temperature of the water bath was double-checked with two thermometers to ensure accuracy.
9. The rack containing the tubes from the ice were removed and placed on the bench top. A tube was opened using a new sterile pipet and 250 µl of LB nutrient broth was added to the tube and reclosed. This was repeated with a new sterile pipet for the other tube. The tube was incubated for 10 minutes at room temperature.
10. The closed tubes were gently flicked with your finger to mix and resuspend the bacteria. A new sterile pipet was used for each tube to transfer 100 µl of the transformation and control suspensions onto the appropriate nutrient ager plates.
11. A new sterile loop is used for each plate. The suspensions were evenly spread around the surface of the LB nutrient ager by skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR. One plate was uncovered and re-covered immediately after spreading the suspension of cells. This minimized contamination.
12. Your plates were stacked and taped together. Your group name and class period were labeled on the bottom of the stack and your stack of plates were placed upside down in the 37°C incubator until the next day. The plates are inverted to prevent condensation on the lid which may drip onto the culture and interfere with your results.