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Team:UQ-Australia/Notebook/Transformation procedure2
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| - | ==Transformation Procedure== | + | ==Transformation Procedure 2== |
1. Thaw competent cells on ice. Place number of required polypropylene tubes on ice. | 1. Thaw competent cells on ice. Place number of required polypropylene tubes on ice. | ||
| - | 2. Gently mix cells, then aliquot 100ul competent cells into chilled polypropylene tubes. | + | 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. |
3. Add 1-10ng of plasmid DNA, moving the pipette through cells while dispersing. Gently tap tubes to mix. | 3. Add 1-10ng of plasmid DNA, moving the pipette through cells while dispersing. Gently tap tubes to mix. | ||
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6. Place on ice for 2 minutes | 6. Place on ice for 2 minutes | ||
| - | 7. Add 900ul of room temperature SOC medium | + | 7. Add 900ul of room temperature SOC medium + glucose |
8. Shake at 225rpm (37'C) for 1 hour | 8. Shake at 225rpm (37'C) for 1 hour | ||
Latest revision as of 05:29, 2 September 2009
Transformation Procedure 2
1. Thaw competent cells on ice. Place number of required polypropylene tubes on ice.
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.
3. Add 1-10ng of plasmid DNA, moving the pipette through cells while dispersing. Gently tap tubes to mix.
4. Incubate cells on ice for 30 minutes
5. Heat-shock cells for 30 seconds in 42'C waterbath. DO NOT SHAKE
6. Place on ice for 2 minutes
7. Add 900ul of room temperature SOC medium + glucose
8. Shake at 225rpm (37'C) for 1 hour
9. During the 1 hour incubation take agar plates out of fridge and incubate in 37'C incubator for 30 minutes
10. Spread 3 agar plates: 50ul, 100ul, 500ul
11. Incubate at 37'C overnight
