Overview
Media used for doing strain construction with the magic marker technology (see references)
Reagents
- 1.7 g YNB without amino acids without ammonium sulfate (Difco, #233520)
- 1 g Monosodium glutamate (L-glutamic acid, monosodium salt MP Biomedicals #194677)
- back to-agar
- glucose
- canavanine (50mg/ml)
- thialysine (50mg/ml)
- clonNat (50mg/ml)
- G418 (142 mg/ml)
- hygromycin (50mg/ml)
For 1L of media, the final composition is:
- 1.7g YNB without amino acids
- 1 g Monosodium glutamate
- 20g bacto-agar (2% bacto-agar)
- 20g glucose (2% glucose)
- canavanine (50mg/L final)
- thialysine (50mg/L final)
- clonNat (50mg/L final)
- G418 (142mg/L final)
- hygromycin (50mg/L final)
Procedure
Combine:
- 1.7g YNB w/o amino acids or ammonium sulfate
- 1g monosodium glutamate
- 20g back to-agar
Bring volume to 900ml with mili-Q water. Autoclave.
Once the solution is cool (~55°C) add 100ml of 20% glucose and drugs as follows:
- Canavanine 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
- Thialysine 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
- G418 1408μL of 142mg/ml stock per 1000ml media (1.408μL stock per ml of media)
- clonNat 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
- hygromycin 6ml of 50mg/ml stock per 1000ml of media (6μL of stock per ml of media)