BTCI -Field Trip Pre-Lab Genetic Transformation, Prep of Competent Cells

1. Steve Harris SPASH-Biotechnical Engineering BTCI-Field TripPre-LabGenetic Transformation, Prep of Competent Cells

2. Competent cell prep protocol • ____Label with your initials one eppendorf tube and working throughout the lab in a cold block. • ____Pipette 1,400µl of näivecells into eppendorf tube. • ____Balance centrifuge, hinge up, centrifuge at 6,000rpm for 2min. • ____Check with black light for fluorescents. Confirm, circle one: [ YES or NO ] • ____Decant supernatant. Remove final volume by pipetting. Leaving a few µl OK. • ____Add 900µl of T8/PMSF neutralization solution to the pellet. This washes and adjust ____the cells pH to a pH of 8. • ____Using a large barrel pipette tip [yellow] re-suspend the pellet followed by pulse vortexing [~20sec.] • ____Balance centrifuge, hinge up, centrifuge at 6,000rpm for 2min. • ____Check with black light for fluorescents. Confirm, circle one: [ YES or NO ] • ____Decant supernatant. Remove final volume by pipetting. Leaving a few µl OK. • ____Add 900µl of T8 buffer to the pellet. This buffers and maintain the cells at a pH 8. • ____Re-suspend the pellet by inversion followed by pulse vortexing [~20sec.] • ____Balance centrifuge, hinge up, centrifuge at 6,000rpm for 2min. • ____Check with black light for fluorescents. Confirm, circle one: [ YES or NO ] • ____Decant supernatant. Remove final volume by pipetting. Leaving a few µl OK. • ____Add 900µl of 100mM CaCl to the pellet to make the cells competent • ____Re-suspend the pellet by inversion followed by pulse vortexing [~20sec.] • ____Return competent cells to your instructor.