McClean:Magic Marker Medias

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(New page: ==Overview== Medias used for doing strain construction with the magic marker technology (see references) ==10x SD/MSG== 1.7 g YNB without amino acids without ammonium sulfate (Difco, #23...)
Current revision (14:36, 11 February 2012) (view source)
(Procedure)
 
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==Overview==
==Overview==
Medias used for doing strain construction with the magic marker technology (see references)
Medias used for doing strain construction with the magic marker technology (see references)
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==10x SD/MSG==
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==Reagents==
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1.7 g YNB without amino acids without ammonium sulfate (Difco, #233520)
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* 1.7 g YNB without amino acids without ammonium sulfate (Difco, #233520)
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1 g Monosodium glutamate (L-glutamic acid, monosodium salt MP BIomedicals #194677)
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* 1 g Monosodium glutamate (L-glutamic acid, monosodium salt MP BIomedicals #194677)
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* bacto-agar
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* glucose
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* canavanine (50mg/ml)
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* thialysine (50mg/ml)
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* clonNat (50mg/ml)
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* G418 (142 mg/ml)
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* hygromycin (50mg/ml)
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===For 1L of media the final composition is:===
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* 1.7g YNB without amino acids
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* 1 g Monosodium glutamate
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* 20g bacto-agar (2% bacto-agar)
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* 20g glucose (2% glucose)
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* canavanine (50mg/L final)
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* thialysine (50mg/L final)
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* clonNat (50mg/L final)
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* G418 (142mg/L final)
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* hygromycin (50mg/L final)
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==Procedure==
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Combine:
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* 1.7g YNB w/o amino acids or ammonium sulfate
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* 1g monosodium glutamate
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* 20g bacto-agar
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Bring volume to 900ml with mili-Q water.  Autoclave.
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Once the solution is cool (~55°C) add 100ml of 20% glucose and drugs as follows:
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* Canavanine 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
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* Thialysine 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
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* G418 1408μL of 142mg/ml stock per 1000ml media (1.408μL stock per ml of media)
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* clonNat 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
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* hygromycin 6ml of 50mg/ml stock per 1000ml of media (6μL of stock per ml of media)
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==Notes==
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Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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'''*[[User:Megan N McClean|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.
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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==References==
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==Contact==
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*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 6 October 2011 (EDT)'''
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
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[[Category:Yeast]]
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[[Category:Protocol]]
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[[Category:Media]]
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Combine YNB and monosodium glutamate in 70mls of dH<sub>2</sub>O.  Dissolve the glutamate and YNB and then bring the final volume to 100mls.  Autoclave.  This solution is 10x.
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Current revision


Contents

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 (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)

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.

References

Contact

or instead, discuss this protocol.

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