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
Serial Dilutions
- This is where the set range of dilution concentrations comes in handy.
- The dilutions for these sets of ranges is a 1:10 dilution. So, a 1-part AHL and 9-part ethyl acetate solution.
- No matter how large the volume of your dilutions, it will always be 1:10.
- For this project, it is a 100uL volume, therefore 10uL will be AHL solution and 90uL ethyl acetate.
- In some cases, the dilution mixture will be a 50uL volume with 5uL being AHL solution and 45uL will ethyl acetate. This is to conserve the longevity of the AHL when doing a single receiver at a time and plating with 2 AHLs.
- For this particular example, however, a 100uL volume and its corresponding 1:10 dilution will be performed.
- 100uL of the 1*10^(-2)M stock solution is pipetted into a tube (for easy access and to prevent contamination of the stock solution)
- 10uL of the stock solution is pipetted into 90uL of Ethyl Acetate to bring the concentration down to 1*10^(-3)M
- Mixing Each Tube:
-
- Pipette up/down
- Vortex
- Spin down (using tabletop centrifuge)
- Continue onto the next dilution
- Steps 2- 3d will be repeated until the final lowest concentration is reached.
- The dilutions will be set up as follows:
- For half dilutions, i.e. 5*10^(-2), 5*10^(-3) it is the same concept except they are 1:1 dilutions instead of 1:10.
- Two ways of doing this includes doing one 1:1 dilution with the stock solution, then doing a 1:10 dilution using the result from the 1:1 dilution. Or it can be done doing a 1:1 dilution for each individual concentration, i.e. 1*10^(-2), 1*10^(-3)
Note: When these solutions are plated, the concentration will be further diluted by 10^-2. For example, 1*10^-2M will be pipetted to its corresponding well on the well-plate, and the AHL final concentration on the well will be 1*10^-4M. This is where the 1*10^-4M to 1*10^-14M concentration range of AHL comes into effect.
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