Team:Arizona State/Notebook

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

2620:

- 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.

Dissolving AHL in Ethyl Acetate

The first thing that must be done is to determine the molecular weight of the AHL, in this case the Lux AHL.
Usually the synthetic AHLs are purchased from Sigma-Aldrich and depending on the volume of what is bought from the company, the milligrams it comes in is important when calculating how much must be dissolved.
In the case of this project, the final stock concentration that was desired was 1*10^(-2)M. In this project, the highest AHL concentration desired was 1*10^(-4)M, but when plated the concentration is diluted down by 10^(-2). Therefore a starting concentrating of 1*10^(-2)M is beneficial.
Then Stoichiometry must be performed to determine the volume in which the AHL in milligrams will be dissolved in, then how much ethyl acetate must be added to get to the desired final stock concentration.

First is to determine the molecular weight and container size the AHL comes in.

213.2 g/mol
10 milligrams of AHL in the container

Then is to determine the desired stock concentration.

This depends upon your range of concentrations, and how high the range is.
Range is: 1*10^(-4)M- 1*10^(-14)M
Therefore stock solution concentration: 1*10^(-2)M

Then determine how large of a stock solution is needed

Since it is difficult to measure out exact milligrams of the AHL and back into a container without losing any grams, a large enough stock solution would be the most beneficial and use the entire AHL in the container.
Desired Volume of Stock Solution: 500uL

Determine Concentration of AHL contained in the stock volume

Here is where stoichiometry comes into play. The milligrams per stock volume must be converted into standard SI molarity.
10mg/500μL * 1g/1*103mg * 1*106L/1μL * 1mol/213.2g = 0.094M
The concentration found is the stock solution concentration needed for dilutions.
Once done with the dilution to 1*10^-2 M, immediately put inside a -80C freezer for long term storage.

With the concentration found above, it will be used in the C1V1=C2V2 equation.

C1 will be the concentration found above.
V1 is the amount of AHL from stock solution into ethyl acetate solution
C2 is the concentration of the stock solution
V2 is the volume of the solution that is going to be further diluted and used in plating
(0.094M)(V1)=(50uL)(1e-2M)
V1=5.32uL AHL

Subtract AHL volume from total volume

This volume of AHL will be used to find the volume of ethyl acetate to dilute the AHL into a 1*10^-2M solution.
So, 500 – 5.32uL = 44.68uL of ethyl acetate

Add these two amounts together in a 1.5mL centrifuge tube

Mixing the Tube:

Pipette up/down
Vortex
Spin down (using tabletop centrifuge)

Immediately place dilution in the -20C freezer for immediate use of plating or for further dilutions.

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