Week 6: Transformation Kit Quick Guide

 In this week, I worked on a transformation kit. 


Materials: 

- two micro test tubes 

- Sterile transfer pipettes

- Ice 

-Sterile loops 

-Bacteria starter plate

- UV lamp 

- media plates

- pGLO plasmid DNA Solution 


Process: 

1. First, label one closed micro test tubes as "+pGLO" and another "-pGLO" and place them in a tube rack. 









2. Using a sterile transfer pipette, transfer 250 ul of transformation solution (CaCl2) 

3. Place the tubes in ice. 

4. Using a sterile loop, pick up a single colony of bacteria from start plate. 

    - pick up loop and put it in the +pGLO tube 

    - spin the loop until the entire colony is dispersed in the transformation solution (no floating chunks) 

    - place the tube back into the ice

    - do again for -pGLO tube using a new sterile loop









5. Examine the pGLO plasmid DNA Solution with the UV Lamp.









6. Immerse a new sterile loop into the plasmid DNA stock tube. 

    - Withdraw a loopful 

7. Mix the loop into the cell suspension of the +pGLO tube. 

    - put it back into ice

    - do not put it in -pGLO 

8. Incubate the tubes in ice for 10 minutes.

9. Label the 4 media plates: 

    - +pGLO LB/amp

    - +pGLO LB/amp/ara

    - -pGLO LB/ amp

    - -pGLO LB

10. Using the tube rack as a holder, transfer both the +pGLO and -pGLO tubes into the water bath. 

    - set at 42 degrees Celsius for exactly 50 seconds 

    - make sure to push the tubes all the way in the rack so the bottom of the tubes won't stick and make contact with the warm water 

    - when done, place tubes back in ice

    - for best transformation results, the change from the ice (0 degrees Celsius) to 42 C and then back to the ice must be rapid. 

    - incubate tube in ice for 2 minutes 

11. Remove the rack containing the tubes from the ice and place on the bench top. 

    - open a tube and using a new pipette, add 250 ul of LB nutrient broth to the tube and reclose it 

    - repeat for the other tube using a new pipette 

    - incubate for 10 minutes at room temperature 

12. Tap the closed tubes with fingers to mix. 

    - using a new pipette, pipette 100 ul of the transformation and control suspensions onto the appropriate plates

13. Using a new pipette for each plate, spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new loop back and forth across the plate surface. 

14. Stack up plates/tape them. Place them upside down in the 37 degree Celsius incubator until the next day. 

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