McClean:Magic Marker Medias

Overview

Medias 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)
• bacto-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 bacto-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)
• Thialysine 1000μL of 50mg/ml stock (per 1000ml media)
• G418 1408μL of 142mg/ml stock (per 1000ml media)
• clonNat 1000μL of 50mg/ml stock (per 1000ml media)
• hygromycin 6μL of 50mg/ml stock per 1ml of media (6mls per 1000mls media)

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

*Megan N McClean: Every time you do this protocol you need to check the drug stock concentrations. For example, the last time the G418 stock was made up the activity of the drug wasn't taken into account, which is why it is a 142mg/ml stock instead of a 200mg/ml stock. Double-check the stock solutions and your final desired concentration before making up the media.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

Contact

• Megan N McClean 14:01, 6 October 2011 (EDT)

or instead, discuss this protocol.