A Dohlman Lab Protocol

Media Recipes

 2X LB (per liter)  
 water  1000 ml
 tryptone  20 g
 yeast extract  10 g
 NaCl  5 g
 pH to 7.0, (~ 52 drops 1N NaOH)  
 for plates (add a stir bar):  
 bacto-agar  15 g
 autoclave & allow to cool to the point that the glass is warm but not burning hot  
 for plasmid selection:  
 ampicillin (or carbenicillin*)
1000 X stock (50 mg/ml)		  1 ml
 *more expensive but more stable, use for plates  

 

LABEL YOUR PLATES USING THE APPROPRIATE COLOR CODE

 2XLB  Blk
 2XLB carb  Blk/Blu
 2XLB kan  Blk/Grn
 SCD (per liter)  
 water  900 ml
 drop-out mix  0.940 g
 yeast nitrogen base w/o amino acids and
w/o ammonium sulfate		  1.7 g
 ammonium sulfate  5 g
 for plates (add a stir bar):  
 bacto-agar  20 g
 NaOH  one pellet
 autoclave & allow to cool to the point that the glass is warm but not burning hot  
 "40X separate mix" (filter sterilized)  25 ml
 20 % dextrose (autoclaved separately)  100 ml

 

LABEL YOUR PLATES USING THE APPROPRIATE COLOR CODE

 SCD-Ura  Red/Grn  SCD-Ura-Leu  Red/Blu/Grn
 SCD-Leu  Red/Blu  SCD-Ura-Trp  Grn/Blu/Blu
 SCD-Trp  Red/Blu/Blu  SCD-Ura-His  Grn/Blu/Blk
 SCD-His  Grn/Blk  SCD-Leu-Trp  Red/Blu/Blk/Blu
 SCD-Ura-Leu-Trp  Red/Red/Red  SCD-Leu-His  Grn/Grn/Blk
 SCD-Ura-Trp-His  Red/Grn/Blu/Blu  SCD-Trp-His  Red/Red/Blu
 SCD-Leu-Trp-His  Blk/Red/Blu/Grn  5FOA  Red/Blk/Grn

 

 YPD (per liter)  900 ml
 water  
 yeast extract  10 g
 peptone  20 g
 for plates (add a stir bar):  
 bacto-agar  20 g
 autoclave  
 20 % dextrose (autoclaved separately)  100 ml
 or  
 20 % galactose  100 ml
 20 % sucrose  10 ml

 

LABEL YOUR PLATES USING THE APPROPRIATE COLOR CODE

 YPD  Red
 YPG/S Red/Red/Blk

 

 

Drop out mix powder

Omit the appropriate amino acid for (URA3, LEU2, HIS3) plasmid selection.
Mix the powder using a coffee grinder.

   Amount (g)  Final conc. (mg/l)
 Arg-HCl  0.4  20
 His-HCl (omit for SCD-His)  0.4  20
 Met  0.4  20
 Uracil (omit for SCD-Ura)  0.4  20
 Ile (omit for SCD-Leu)  0.6  30
 Leu (omit for SCD-Leu)  0.6  30
 Lys-HCl  0.6  30
 Tyr  0.6  30
 Adenine-HCl  0.8  40
 Phe  1.0  50
 Glu  2.0  100
 Val  3.0  150
 Ser  8.0  400
 Add 0.940 g to 1 liter of media & autoclave    
 40X Separate mix liquid    
   Amount (g)  Final conc. (mg/l)
 Trp (omit for SCD-Trp)  0.8  40
 Asp  2.0  100
 Thr  4.0  200

Dissolve in 500 ml water, filter sterilize, and add 25 ml per liter of media
(after autoclaving & cooling)


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