A Dohlman Lab Protocol

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)