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<center>These protocols are used to screen bacterial plates for [[beta-galactosidase]] activity using X-
<center>These protocols are used to screen bacterial plates for [[beta-galactosidase]] activity using X-(aka blue white screening). <br> Each of these protocols has its advantages and disadvantages so be sure to check which one works best for you.</center>
Revision as of 16:29, 22 March 2011
Making X-gal Plates
Each of these protocols has its advantages and disadvantages so be sure to check which one works best for you.
- X-gal Stock solution = 100mg/ml in dimethyl formamaide (DMF)
- Media of your choice
- Sterile water
Making X-gal plates
- This method is used if you won't be keeping the plates more than a week. The media used should have a neutral pH.
- Colonies may take up to 36 hours to develop on these plates, but all positive colonies will be evenly colored.
- Make the media according to your normal plate recipe and autoclave.
- Let the media cool in a 60°C water bath.
- Add X-gal stock solution at a ratio of 2μL per mL media (also add any antibiotic).
- Swirl gently and pour immediately.
- Once solid keep out of light.
Spread onto LB plates
- This method is used to turn any kind of plate into an X-gal plate. The media should have a neutral pH.
- 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.
- Dilute the X-gal stock 1 to 1 with sterile water.
- 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.
- Evenly distribute the solution onto the plate using a spreader.
- Let plate dry for 30mins at 37°C.
- This is used if: your media has a non-neutral pH, you forgot to one of the other two protocols, some undergrad in your lab mislabeled the plates.
- Color should appear on fully developed colonies within 8 hours.
- 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).
- Let the molten agar cool in a 60°C water bath.
- Add 2μL X-gal stock for each mL of molten agar (e.g. 200μL for 1 plates).
- Pour 10 ml over each plate to be assayed.
- Let solidify
- Incubate plates in a dark place