Flowchart
Label two microtubes (+) and (-)
(ONE TUBE IS GOING TO HAVE A PLAID ANF THE OTHER ONE ISINT)*
(ONE TUBE IS GOING TO HAVE A PLAID ANF THE OTHER ONE ISINT)*
DRAW AND LABEL 3 PLATES *(MAKE A LINE DOWN THE MIDDLE OF PLATES 1 AND 2 NT 3)**
ON ONE HALF PLACE P+
ON THE OTHER HAL PLACE P -
PLATE 3 MARK P+
-Use a micropipette ( set it to 050:)
-Add 50 ul of cells solution to both (+) and (-) tubes
With the smaller volume micropipette (set it to 10)
*use a sterile pipette tip*
Add 10 ul ofile://localhost/Users/student/Desktop/8934302.jpg
d rpaRA plasmid to the (+) tube only
-Add 50 ul of cells solution to both (+) and (-) tubes
With the smaller volume micropipette (set it to 10)
*use a sterile pipette tip*
Add 10 ul ofile://localhost/Users/student/Desktop/8934302.jpg
d rpaRA plasmid to the (+) tube only
Incubate both (+) and (-) tubes on ice (*for 10 min*)
*(to reproduce the bacteria and to make pressure differ*)
START: 242 END: 252
*(to reproduce the bacteria and to make pressure differ*)
START: 242 END: 252
Take the tubes in ice water cup to 42C in a hot water bath
*(make sure its 42C)* *( trasfer the tubes from ice to the hot water
*(for 45-50 seconds*)
*(immediately return the tubes to ice for 2 minutes.
start: 252 end :2:53
*(make sure its 42C)* *( trasfer the tubes from ice to the hot water
*(for 45-50 seconds*)
*(immediately return the tubes to ice for 2 minutes.
start: 252 end :2:53
Place (+) and (-) tubes in a beaker/cup at room temperature
*(THIS NORMALIZES BACTERIA*)
add 150UL LB broth to both tubes*)
THEN MIX IN VORTEX FOR 2 MINUTES
*(THIS NORMALIZES BACTERIA*)
add 150UL LB broth to both tubes*)
THEN MIX IN VORTEX FOR 2 MINUTES
Use micropipette with steril tip for (-) loading and a new steril tip to load (+) tube *(50 ul onto plates 1 and 2 *( be sure to place on (+) side only*)
-and then 50 ul ON PLATE 3
-and then 50 ul ON PLATE 3
USE INACULATION LOOP TO GENTLY SPREAD THE (-) SIDE OF plate 1 and 2
then use another inoculation loop and gently spread the (+) sides of plates 1 2 and 3
then use another inoculation loop and gently spread the (+) sides of plates 1 2 and 3
Cover and stack each plate *( after 15 minutes invert plates (agar side up)
*( tape and label with teams name*)
(* incubate at 37C for 24 hours*)
*( tape and label with teams name*)
(* incubate at 37C for 24 hours*)