User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/07

Transformation protocol

Transformation of four strains of competent cell: DH5α, JMV 101, MACH1 and XL-1BLUE with plasmid BBa_Kate_GFP

1. Take out DH5α strainfrom the -80°C freezer and place it in the ice bucket. Repeat this for other strains.
2. Take out plasmid BBa_Kate_GFP from the -20°C freezer and plave it in the ice bucket.
3. Take out the Soc from the -20°C freezer and place it in the incubater at 37°C.
4. Add 1μL of the plasmid and transfer into each strains. Mix it very well. Note: This need to be completed in front of the flame.
5. Put the tubes back into the ice bucket for 5 min.
6. The tubes is placed in waterbath at 42°C for 1 min exactly.
7. Add 250μL of Soc into each tubes and vortex.
8. The tubes is then placed in the shaker for about 1 hr.
9. Take out agar plates with Ampicillin and place it in the incubater at 37°C for 5 min upsidedown with the lid open slightly.
10. Take out the tubes from the shaker and the plates from the incubater.
11. Transfer the strains into each plates by pouring onto the surface of the plates.
12. Add 8-10 glass beads into the plate and shake the plates side to side and all around.
13. Remove the glass beads by tapping on one side of the plate.
14. The plates are placed in the incubater at 32°C for overnight (16hrs).

Inoculation

Four 10 ml bottles of LB Broth with Ampicillin were inoculated and grown overnight from colonies generated from various strains transformed with plasmid BBa_Kate_GFP

1. Four 10 ml LB broth bottles are required for four 200 ml plates that are transformed overnight with plasmid BBa_Kate_GFP.
2. The colonies are removed from each plates using----- and inoculated into each 10 ml broth tube.
3. The bottles are labelled and placed in the shaker for 16 hrs, overnight.