Team:UQ-Australia/Notebook/Colony pick procedure

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==Transformation Procedure 2==
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==Colony Picking Procedure==
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1. Thaw competent cells on ice. Place number of required polypropylene tubes on ice.
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Micro-culture
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2. Gently mix cells, then aliquot 100ul competent cells into chilled polypropylene tubes. Add 1.7 ul of B-mercaptoethanol. Gently stir every 2 minutes for 10 minutes.
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3ml of LB Broth
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3ml of Antibiotic (depends on the antibiotic resistance gene on the plasmid)
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3. Add 1-10ng of plasmid DNA, moving the pipette through cells while dispersing. Gently tap tubes to mix.
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1. Touch a single colony with a pipette tip and place the entire tip into the tube
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4. Incubate cells on ice for 30 minutes
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2. Repeat 5 times or for as many colonies which are present
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5. Heat-shock cells for 30 seconds in 42'C waterbath. DO NOT SHAKE
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3. Place in incubator/shaker overnight at 37'C
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6. Place on ice for 2 minutes
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7. Add 900ul of room temperature SOC medium + glucose
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8. Shake at 225rpm (37'C) for 1 hour
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9. During the 1 hour incubation take agar plates out of fridge and incubate in 37'C incubator for 30 minutes
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10. Spread 3 agar plates: 50ul, 100ul, 500ul
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11. Incubate at 37'C overnight
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Latest revision as of 05:47, 2 September 2009

Colony Picking Procedure

Micro-culture

3ml of LB Broth 3ml of Antibiotic (depends on the antibiotic resistance gene on the plasmid)

1. Touch a single colony with a pipette tip and place the entire tip into the tube

2. Repeat 5 times or for as many colonies which are present

3. Place in incubator/shaker overnight at 37'C

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