Team:Austin UTexas/Protocols


HS Media
Introduction
This is the recipe to make 1 Liter of HS (High Salt) medium.
Procedure

  1. 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)
  2. Add distilled water to reach final volume
  3. Autoclave the solution
  4. 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

  1. Add the following compounds
    • 10 g of Yeast Extract
    • 20 g of Peptone
    • 16 g of Agar (if making solid media)
  2. Add distilled water to reach final volume of 1 L
  3. Autoclave the solution
  4. 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

  1. Add 400 mL to an empty bottle
  2. 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)
  3. Autoclave the solution


4x Sucrose Solution
Introduction
This is the recipe to make 472.5 mL of 4x Sucrose Solution
Procedure

  1. Add 1 cup of Sucrose to a bottle
  2. 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
  3. Autoclave the solution


Conjugation With DAP
Introduction
This is the protocol for conjgating a plasmid using a DAP dependent donor strain and a targeted recipient strain.
Materials

  1. .3 mM Diaminopimelic Acid (DAP)
  2. DAP Auxotroph strain with a plasmid
    • This plasmid should have an antibiotic resistant gene, an origin of transfer and a gene of interest.
  3. Recipient Strain
  4. LB+DAP Plates
  5. LB+Antibiotic Plates
    • The antibiotic should be the one that plasmid has a resistance to.
  6. Phosphate Buffered Saline (PBS)


Procedure

  1. 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.
  2. Wash the donor and recipient cells