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Dorman:P22 transduction: Difference between revisions
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'''Day 0''' | '''Day 0''' | ||
#Grow up the recipient strain overnight in L. | #Grow up the recipient strain overnight in L. | ||
'''Day 1''' | '''Day 1''' | ||
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* 100 μL of recipient strain (no phage control- confirms that there are no mutants in the recipient population) | * 100 μL of recipient strain (no phage control- confirms that there are no mutants in the recipient population) | ||
* 100 μL of P22 lysate (no recipent control- confirms that the lysate is not contaminated with bacteria.) | * 100 μL of P22 lysate (no recipent control- confirms that the lysate is not contaminated with bacteria.) | ||
#Incubate these mixtures at 37°C (no shaking necessary) for 1 hour. | #Incubate these mixtures at 37°C (no shaking necessary) for 1 hour. | ||
#Plate the entire volume of the transduction mixtures onto L agar plates with appropriate antibiotics. | #Plate the entire volume of the transduction mixtures onto L agar plates with appropriate antibiotics. | ||
'''Day 2''' | '''Day 2''' | ||
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#Streak a desired number of colonies onto green agar plates. I typically pick four for routine strain construction. | #Streak a desired number of colonies onto green agar plates. I typically pick four for routine strain construction. | ||
'''Day 3''' | '''Day 3''' | ||
Examine your green plates. I typically pick one pale colony of each plate and streak again on green agar. | Examine your green plates. I typically pick one pale colony of each plate and streak again on green agar. | ||
'''Day 4''' | '''Day 4''' | ||
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(We get solidified green base from the prep room in 100 mL bottles.) | (We get solidified green base from the prep room in 100 mL bottles.) | ||
#To make the plates, at 62.5 mg Alizarin yellow (on Dan's bench) to the 100 mL bottle of green base, and place the agar in the steamer. | #To make the plates, at 62.5 mg Alizarin yellow (on Dan's bench) to the 100 mL bottle of green base, and place the agar in the steamer. | ||
#When the 100mL bottle comes out of the steamer and has been cooled in the 55° bath, add 0.33 mL of 2% Aliline blue (on Dan's bench), 4.2 mL of 20% glucose, and appropriate antibiotics. | #When the 100mL bottle comes out of the steamer and has been cooled in the 55° bath, add 0.33 mL of 2% Aliline blue (on Dan's bench), 4.2 mL of 20% glucose, and appropriate antibiotics. | ||
