Beta-galactosidase Screen

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(Agar Overlay)
Current revision (13:52, 14 September 2011) (view source)
 
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Making X-gal Plates
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<center>'''Making X-gal Plates'''</center>
   
   
==Introduction==
==Introduction==
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==Methods==
==Methods==
===Making X-gal plates===
===Making X-gal plates===
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*This method is used if you won't be keeping the plates more than a week.  The media used should have a neutral pH.
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*This method is used if: you won't be keeping the plates more than a week, and the media has a neutral pH.
*Colonies may take up to 36 hours to develop on these plates, but all positive colonies will be evenly colored.
*Colonies may take up to 36 hours to develop on these plates, but all positive colonies will be evenly colored.
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#Swirl gently and pour immediately.
#Swirl gently and pour immediately.
#Once solid keep out of light.
#Once solid keep out of light.
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===Spread onto LB plates===
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===Spread onto plates===
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*This method is used to turn any kind of plate into an X-gal plate.  The media should have a neutral pH.
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*This method is used if: you want to turn an existing plate into an X-gal plate, and the media has a neutral pH.
*Colonies should develop color overnight, but due to uneven distribution of X-gal, all positive colonies may not be evenly colored.
*Colonies should develop color overnight, but due to uneven distribution of X-gal, all positive colonies may not be evenly colored.
#Take a pre-poured plate and make sure it is dry (i.e. no condensation on the media) and solid.
#Take a pre-poured plate and make sure it is dry (i.e. no condensation on the media) and solid.
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===Agar Overlay===
===Agar Overlay===
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*This is used if: your media has a non-neutral pH, you forgot to do one of the other two protocols, or some undergrad in your lab mislabeled the plates.  
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*This method is used if: your media has a non-neutral pH, you forgot to do one of the other two protocols, or some undergrad in your lab mislabeled the plates.  
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*Color should appear on fully developed colonies within 8 hours.
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*Color should appear on fully developed colonies in 2-8 hours.
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#Microwave 10ml of water with 0.15g agar for each plate to be screened (e.g. for 10 plates its 100ml water and 1.5g agar).
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#Microwave 10ml of water with 0.15g agar for each plate to be screened (e.g. for 10 plates it's 100ml water and 1.5g agar).
#Let the molten agar cool in a 60°C water bath.
#Let the molten agar cool in a 60°C water bath.
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#Add 2μL X-gal stock for each mL of molten agar (e.g. 200μL for 1 plates).
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#Add 2μL X-gal stock for each mL of molten agar (e.g. 200μL for 10 plates).
#Pour 10 ml over each plate to be assayed.
#Pour 10 ml over each plate to be assayed.
#Let solidify
#Let solidify

Current revision

Making X-gal Plates

Contents

Introduction

These protocols are used to screen bacterial plates for beta-galactosidase activity using X-Gal (aka blue white screening).
Each of these protocols has its advantages and disadvantages so be sure to check which one works best for you.

Materials

  • X-gal Stock solution = 100mg/ml in dimethyl formamaide (DMF)
  • Agar
  • Media of your choice
  • Sterile water

Methods

Making X-gal plates

  • This method is used if: you won't be keeping the plates more than a week, and the media has a neutral pH.
  • Colonies may take up to 36 hours to develop on these plates, but all positive colonies will be evenly colored.
  1. Make the media according to your normal plate recipe and autoclave.
  2. Let the media cool in a 60°C water bath.
  3. Add X-gal stock solution at a ratio of 2μL per mL media (also add any antibiotic).
  4. Swirl gently and pour immediately.
  5. Once solid keep out of light.

Spread onto plates

  • This method is used if: you want to turn an existing plate into an X-gal plate, and the media has a neutral pH.
  • Colonies should develop color overnight, but due to uneven distribution of X-gal, all positive colonies may not be evenly colored.
  1. Take a pre-poured plate and make sure it is dry (i.e. no condensation on the media) and solid.
  2. Dilute the X-gal stock 1 to 1 with sterile water.
  3. Pipette 50μL of this dilution onto the plate (If your plate has condensation on it skip the dilution and pipette 25μL of X-gal stock.
  4. Evenly distribute the solution onto the plate using a spreader.
  5. Let plate dry for 30mins at 37°C.

Agar Overlay

  • This method is used if: your media has a non-neutral pH, you forgot to do one of the other two protocols, or some undergrad in your lab mislabeled the plates.
  • Color should appear on fully developed colonies in 2-8 hours.
  1. Microwave 10ml of water with 0.15g agar for each plate to be screened (e.g. for 10 plates it's 100ml water and 1.5g agar).
  2. Let the molten agar cool in a 60°C water bath.
  3. Add 2μL X-gal stock for each mL of molten agar (e.g. 200μL for 10 plates).
  4. Pour 10 ml over each plate to be assayed.
  5. Let solidify
  6. Incubate plates in a dark place

See also

References

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