Team:Brown-Stanford/Lab/Protocols/NEB 5-alpha

From 2011.igem.org

(Difference between revisions)

Revision as of 06:36, 28 September 2011

Overview

For C2987H, 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.

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.

@@ 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])
+==Overview==
+For C2987H, 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.
 ==Solutions needed==