Feb 15, 2022
Version 3
High Efficiency Transformation Protocol (C2987H) V.3
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987
Protocol Citation: New England Biolabs 2022. High Efficiency Transformation Protocol (C2987H). protocols.io https://dx.doi.org/10.17504/protocols.io.bddti26nVersion 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: February 15, 2022
Protocol Integer ID: 33939
Keywords: Transformation , Bacteria, C2987H,
Abstract
This is the protocol for C2987H cells. If you are using the C2987I cells, please refer to this protocol.
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
| A | B | |
| 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 (NEB #T1030) or phenol/chloroform extraction and ethanol precipitation should be added.
Materials
MATERIALS
SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S
NEB 5-alpha Competent E. coli cellsNew England BiolabsCatalog #C2987H
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
For this protocol, perform steps 1-8 in the tube provided.
Thaw a tube of NEB 5-alpha 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 SOC into the mixture.
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. 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.
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 SOC.
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 .