Media Recipes
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)