Kombucha Protocol

'''HS Media'''
'''Introduction'''
This is the recipe to make 1 Liter of HS (High Salt) medium.
'''Procedure''' # Add the following coumpound #*5 g of Yeast Extract #*5 g of Peptone #*2.7 g of Sodium Phosphate, dibasic anhydrous (Na₂HPO₄) #*1.5 g of Citric Acid #*16 g of Agar (if making solid media) #Add distilled water to reach final volume #Autoclave the solution # Add 50 mL of Sterile 40% (w/v) glucose after the solution has been autoclaved '''DO NOT ADD GLUCOSE PRIOR TO AUTOCLAVING THE SOLUTION TO AVOID CREATING TOXIC BYPRODUCTS''' '''YPD Media'''
'''Introduction'''
This is the recipe to make 1 Liter of YPD medium which is a complete medium for yeast growth.
'''Procedure''' # Add the following compounds #*10 g of Yeast Extract #*20 g of Peptone #*16 g of Agar (if making solid media) #Add distilled water to reach final volume of 1 L #Autoclave the solution # Add 50 mL of Sterile 40% (w/v) glucose after the solution is autoclaved '''DO NOT ADD GLUCOSE PRIOR TO AUTOCLAVING THE SOLUTION TO AVOID CREATING TOXIC BYPRODUCTS''' '''PBS (Phosphate Buffered Saline) Solution'''
'''Introduction'''
This is the recipe to make 500 mL of PBS which is used in a variety of procedures.
'''Procedure''' # Add 400 mL to an empty bottle # Add and dissolve each of the following compounds in the water: #* 4.0 g of Sodium Chloride (NaCl) #* 0.1 g of Potassium Chloride (KCl) #* 0.72 g of Sodium Phosphate, dibasic anhydrous (Na₂HPO₄) #* 0.12 g of Potassium Phosphate, monobasic (KH₂PO₄) # Autoclave the solution '''4x Sucrose Solution'''
'''Introduction'''
This is the recipe to make 472.5 mL of 4x Sucrose Solution
'''Procedure''' # Add 1 cup of Sucrose to a bottle # Fill the bottle with distilled water until it reaches a final volume of 472.5 mL #*As the bottle is being filled, swirl the contents so that the sucrose can begin to dissolve # Autoclave the solution '''Conjugation With DAP'''
'''Introduction'''
This is the protocol for conjugating a plasmid using a DAP dependent donor strain and a targeted recipient strain.
'''Materials''' #.3 mM Diaminopimelic Acid (DAP) #DAP Auxotroph strain with a plasmid #*This plasmid should have an antibiotic resistant gene, an origin of transfer and a gene of interest. #Recipient Strain #LB+DAP Plates #LB+Antibiotic Plates #*The antibiotic should be the one that plasmid has a resistance to. #Phosphate Buffered Saline (PBS)
'''Procedure''' #Grow up the donor and recipient strains #*For the donor cells, inoculate liquid LB+antibiotic+DAP (the one that the plasmid is resistant to). Depending on the amount of liquid media used, the volume should be divided by 1000 and then multiplied by 3 in order to determine the amount of DAP used. Ex. 5 mL of media will need 15 µL of DAP. #*For the recipient cells, grow them in the required liquid media and an antibiotic if needed. #Wash the donor and recipient cells #*After sufficient growth, transfer 1 mL of each strain into a microcentrifuge tube. #*Centrifuge the tubes at 3,000 RPM for 1 minute, for a pellet to form. #*After centrifugation, decant the supernatant, add 1 mL of PBS to the tube, and resuspend the pellets by vortexing it. #*Centrifuge these tubes again at 3,000 RPM for 1 minute and 30 seconds. #*Decant the supernatant and resuspend the pellets with 1 mL of PBS. #Combine the cells into a microcentrifuge tube #*Take an OD600 for both cell types and make a 1:1 ratio of donor and recipient cells #*Combine 50 µL of each cell type at a 1:1 ratio into a microcentrifuge tube #*Spin down the solution for 3 seconds in a mini centrifuge. #Plate the solutions and incubate #*Transfer 100 µL of the solution onto a LB+DAP plate. Do not spread. You can make 5 different conjugations on a plate by dividing up the plate. #*Incubate these plates overnight #Scrape up the growth #*After incubation, scrape up the growth with an inoculating loop and resuspend the colonies in 1 mL of PBS. #Wash the solution #*Spin down the microfuge tubes at 3,000 RPM for 1 minute and 30 seconds. #*Decant the supernatant. #*Repeat #*Resuspend the mixture of cells in 1 mL of PBS #Plate the mixture of cells #*Plate 50 µL of each mixture of cells on a LB+the antibiotic the plasmid is resistant to. However, this plate can't have DAP. #*Make sure to plate 1 mixture per plate and spread it. #*Incubate overnight #Pick the transconjugants #*With a sterile loop, pick an isolated colony that appears to have been successfully conjugated. #*Streak out this colony onto another plate of the same kind (LB+Antibiotic). #*Incubate this plate for 24 hours. #Create a liquid culture #*Pick an isolated colony with a sterile loop and inoculate it into 5 mL of LB + 5 µL of the antibiotic. #*Place these tubes into a shaker incubator for 24 hours. #Freeze down the isolate #*Pipette 1.6 mL of the liquid culture into a cryovial. #*Add 400 µL of 100% DMSO. #*Turn upside down and back a few times to mix. #*Place into a -80℃ freezer for future use. '''LB+DAP Agar Plate Recipe'''
'''Introduction'''
This is the protocol for making LB+DAP agar plates to be used during conjugation.
'''Materials''' #LB Agar #Diaminopimelic Acid (DAP) #Sterile Petri Dishes #50 mL Falcon Tube
'''Procedure''' #Obtain a bottle of sterile, LB Agar. Heat the bottle in a microwave at half or medium power for approximately 5-8 minutes or until completely melted. #After allowing the LB agar to cool down slightly, using sterile technique, gently pour 25 mL of LB agar into the Falcon Tube. #Add 75 µL of DAP to the LB agar in the Falcon Tube. #With the lid tightly secured on the tube, gently invert the tube in order to mix the LB Agar and DAP sufficiently. #Lastly, pour the entire mixture into the plate and allow all of the plates to dry/solidify before moving or storing them.