Sep 08, 2022
High Efficiency Transformation
- 1New England Biolabs
Protocol Citation: New England Biolabs 2022. High Efficiency Transformation . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2bdzg5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 06, 2022
Last Modified: July 11, 2023
Protocol Integer ID: 69627
Abstract
Protocol for high efficiency heat shock transformation of competent E. coli cells.
Source: New England Biolabs
Materials
SOC Outgrowth Medium - 100 ml (New England Biolabs Catalog #B9020S)
Thaw a tube of competent E. coli cells on ice for 00:10:00 . Mix gently and carefully pipette 50 µL of cells into a transformation tube on ice.
10m
Add 1 µL containing 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 00:30:00 . Do not mix.
30m
Heat shock at exactly 42 °C for exactly 00:00:30 . Do not mix.
30s
Place on ice for 00:05:00 . Do not mix.
5m
Pipette 950 µL of room temperature SOC into the mixture.
Place at 37 °C for 01:00:00 . Shake vigorously (250 rpm ) or rotate.
1h
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 24:00:00 or 25 °C for 48 hours.
1d