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Team:Brown-Stanford/Lab/Protocols/NEB 5-alpha
From 2011.igem.org
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==Overview== | ==Overview== | ||
| - | For C2987H, perform steps 1-7 in the tube provided. | + | For C2987H (5-alpha competent cells), perform steps 1-7 in the tube provided. |
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==Protocol== | ==Protocol== | ||
Revision as of 06:49, 28 September 2011
NEB 5-alpha High-Efficiency Transformation
(Taken from [http://www.neb.com/nebecomm/products/protocol119.asp the New England BioLabs Protocol])
Overview
For C2987H (5-alpha competent cells), perform steps 1-7 in the tube provided.
Protocol
- For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
- For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
- Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
- Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
