"
Team:Brown-Stanford/Lab/Protocols/NEB 5-alpha
From 2011.igem.org
(→'"NEB 5-alpha High-Efficiency Transformation) |
(→"'NEB 5-alpha High-Efficiency Transformation) |
||
| Line 1: | Line 1: | ||
| - | == | + | === NEB 5-alpha High-Efficiency Transformation === |
Taken from [http://www.neb.com/nebecomm/products/protocol119.asp the New England BioLabs Protocol] | Taken from [http://www.neb.com/nebecomm/products/protocol119.asp the New England BioLabs Protocol] | ||
| - | + | ==Solutions needed== | |
Revision as of 06:24, 28 September 2011
NEB 5-alpha High-Efficiency Transformation
Taken from the New England BioLabs Protocol
Solutions needed
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.
