Apr 11, 2022
Version 6
High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019) V.6
- 1New England Biolabs
External link: https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c3019
Protocol Citation: New England Biolabs 2022. High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019). protocols.io https://dx.doi.org/10.17504/protocols.io.zewov12ogr24/v6Version created by New England Biolabs
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: March 08, 2020
Last Modified: April 11, 2022
Protocol Integer ID: 33941
Keywords: Transformation , Bacterial, C3019I, C3019, C3019H,
Abstract
This is the High Efficiency Transformation Protocol for C3019H and C3019I cells.
Guidelines
Transformation Protocol Variables
Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.
Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.
Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
DNA Contaminants to Avoid
| Contaminant | Removal Method | |
| Detergents | Ethanol precipitate | |
| Phenol | Extract with chloroform and ethanol precipitate | |
| Ethanol or Isopropanol | Dry pellet before resuspending | |
| PEG* | Column purify or phenol/chloroform extract and ethanol precipitate | |
| DNA binding proteins (e.g. Ligase) | Column purify or phenol/chloroform extract and ethanol precipitate |
* Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.
Materials
MATERIALS
NEB 10-beta Competent E.coli (High Efficiency) - 20x0.05 mlNew England BiolabsCatalog #C3019H
NEB 10-beta Competent E.coli (High Efficiency) - 6x0.2 mlNew England BiolabsCatalog #C3019I
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Please select whether you have C3019H or C3019I cells.
C3019H13 steps
Thaw a tube of NEB 10-beta Competent E. coli cells On ice for 00:10:00 .
Note
Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
Add 1 µL -5 µL containing 1 pg -100 ng 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.
Note
For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.
Heat shock at exactly 42 °C for exactly 00:00:30 . Do not mix.
Note
Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.
Place On ice for 00:05:00 . Do not mix.
Pipette 950 µL room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture.
Note
NEB 10-beta/Stable Outgrowth Medium gives 2-fold higher transformation efficiency than LB medium.
Place at 37 °C for 01:00:00 , shaking vigorously (250 rpm ) or rotating.
Note
Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. Incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
Warm selection plates to 37 °C .
Note
Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
Mix the cells thoroughly by flicking the tube and inverting.
Perform several 10-fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.
Note
Please note that NEB no longer recommends SOC for use in this protocol. NEB 10-beta/Stable Outgrowth Medium is now provided with NEB 10-beta Competent E.coli and available also as a stand-alone product.
Spread 50 µL -100 µL of each dilution onto a selection plate.
Incubate Overnight at 37 °C .
Note
Alternatively, incubate at 30 °C for 24-36 hours or 25 °C for 48:00:00 .