Arizona_State
Notebook: Brianna Lopez
Transformation of Negative Sender and LuxR
- These colonies will be used for overnight liquid culture growth, which will be used in 96-well plating for overnight growth curves.
- Transformation of a negative sender, and 2620 vector.
- Concentration of 2620 & Neg Sender:
-2620 = 135 ng/uL
-Neg Sender = 461 ng/uL (will have to do 1:10 dilution)
- Calculation Calculation to get concentration of 60 ng/uL each:
- Negative Sender:
- 461 ng/ul (1uL into 9uL H20) = 46.1 ng/uL
- 46.1 ng/uL * x uL = 60 ng
- x = 1.3 uL
- 135 ng/uL * x uL = 60 ng
- x = .44 uL
Protocol:
Materials:
- Ice bucket
- 2620
- Negative Sender
- DI H20
Dilution Procedure:
- One 1.5mL tube was used to perform a dilution, and labeled dilution tube.
- 9uL of DI H20 was pipetted into the dilution tube.
- 1uL of negative sender was taken out of stock and pipetted into the dilution tube.
- The volume was then pipetted up and down to mix and dilute the solution.
Transformation Procedure:
- One tube was labeled 2620, the other negative sender
- 1.3uL of negative sender was pipetted from the dilution tube into the negative sender tube
- 0.44uL of 2620 was pipetted from 2620 stock tube into the 2620 tube.
- Tubes were then placed on ice for 2-5 minutes
- The tubes were then placed on the hot plate of 42C for exactly 45 seconds then immediately placed on ice for 5 minutes.
- 300uL of SOC was added to each tube
- It was then placed in the 37C shaking incubator for 20-30 minutes
- The tubes were then spun down at 3000 rpm for 1 minute
- The liquid in the tubes were gently tapped out into liquid waste
- 100uL of SOC was added to the tubes and gently pipetted up and down to resuspend the cells
- 100uL of each tube were then plated onto LB/AMP plates
- Then placed in 37̊C still incubator over night
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