McClean:Magic Marker Medias

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(Procedure)
 
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==Reagents==
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==10x SD/MSG==
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* 1.7 g YNB without amino acids without ammonium sulfate (Difco, #233520)
* 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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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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===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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==Materials==
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==Procedure==
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*α1-Mating Factor acetate salt (Sigma T6901-1mg)
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Combine:
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*DMSO (Fluka 41639, Ultra for molecular biology; stored in the Flammables cabinet)
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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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==Procedure==
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* Canavanine 1000μL of 50mg/ml stock per 1000ml media (1μL stock per ml of media)
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Shake the new vial of α-factor to get all of the power on the bottom of the vial. Remove the lid and pipet 1ml of DMSO into the vial of α-factor from Sigma.  Pipet up and down and put the lid back on an swirl to dissolve all of the alpha-factor into the DMSO.  Then remove the liquid and aliquot it in 100μL eppendorfs into individual eppendorfs and store these in your own -20°C box.  ''Keep track of the lot # of the pheromone both with your stock solutions and when you do your experiments.''
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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)
==Notes==
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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]]''': Make sure to keep track of lot numbers (write it down in your notebook, on your stock solutions, and each time you do an experiment with α-factor).  '''Really'''.  Please do this!
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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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'''*[[User:Megan N McClean|Megan N McClean]]''': I generally find it useful to make a series of 1:10 dilutions of the α-factor (1mg/mL, 100μg/mL, 10μg/mL, 1μg/mL) when I first make the stock, as these are 1000x the concentrations I generally start out with for most experiments (so that I can add 1μL of stock solution per ml of media)I usually make 900μL of all of these dilute stocks (ie, start with 100μL of 1mg/ml stock +900μL of DMSO for the 100μg/mL stock, from that take 100μL and add it to another 900μL of DMSO for the 10μg/mL, and then once more for the 1μg/mL stock)I then divide the 900μL into 100μL aliquots so that I don't have to freeze/thaw the entire stock too often.
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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.
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==
==Contact==
==Contact==
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[[Category:Yeast]]
[[Category:Yeast]]
[[Category:Protocol]]
[[Category:Protocol]]
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[[Category:Media]]
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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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