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A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride
Published: Nov 5, 2017 DOI: 10.21769/BioProtoc.2590 Views: 19714
Edited by: Dennis Nürnberg Reviewed by: Christian SailerMichael Tscherner
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Abstract
This protocol describes a quick and efficient method to make competent E. coli cells for transformation using rubidium chloride. Commercial competent cells are expensive and this protocol provides a cheaper alternative to them.
Keywords: Competent cells
E. coli
Transformation efficiency
TOP10
DH5α
Background
The success of gene cloning is highly dependent on the transformation efficiency of bacterial cells. The efficiency can be artificially improved by treating the cells with chemicals or electric pulses. Several protocols are available to prepare competent E. coli cells, however, they are usually long, laborious, and show inconsistency in competence. The protocol by Green and Rogers (2013) overcomes these downsides and allows the preparation of highly competent cells (~106-108 CFU/µg DNA). While other protocols require cells to be grown at low temperature (19-22 °C), this protocol involves growing cells at 37 °C. Thus, the cells grow faster and reach log phase within 4 h as compared to 18-24 h. This protocol is highly reproducible.
Materials and Reagents
- 25 mm, 0.2 µm syringe filter PES (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 725-2520 )
- 5 ml syringe (Fisher Scientific, catalog number: 14-829-45)
Manufacturer: BD, catalog number: 309646 . - 1.5 ml Eppendorf tubes (Corning, Costar®, catalog number: 3207 )
- 100 mm Petri dish (Corning, Falcon®, catalog number: 351029 )
- Pipettes tips
- 10 ml culture tubes (VWR, catalog number: 60818-725 )
- Spreaders (Fisher Scientific, catalog number: 14-665-230 )
- 250 ml centrifuge bottles (Sigma-Aldrich, catalog number: Z353736)
Manufacturer: Thermo Fisher Scientific, catalog number: 3141-0250 . - E. coli strain (TOP10 or DH5α)
- Control plasmid (e.g., pUC19)
- LB broth (Fisher Scientific, catalog number: BP9723-500 )
- LB agar (BD, catalog number: 244510 )
- Ice
- Liquid nitrogen
- Rubidium chloride (RbCl) (Sigma-Aldrich, catalog number: R2252 )
- Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 203734 )
- Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
- Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C8106 )
- Glycerol (Fisher Scientific, catalog number: BP229-4 )
- MOPS (Fisher Scientific, catalog number: BP308-100 )
- Acetic acid (Fisher Scientific, catalog number: A38-212 )
- Sodium hydroxide (Fisher Scientific, catalog number: S318-1 )
- Vector specific Antibiotics
- Buffer I (see Recipes)
- Buffer II (see Recipes)
Equipment
- Pipettes (P1000 and P200)
- 1 L flasks (Fisher Scientific, catalog number: 10-040K)
Manufacturer: Corning, catalog number: C49801L . - Spectrophotometer (Beckman Coulter, model: DU-640 , catalog number: 8043-30-1090)
- Water bath (Marshall Scientific, model: Precision 181 )
- 37 °C incubator and shaker (Thermo Fisher Scientific, model: MaxQTM 4000 , catalog number: SHKE4000-1CE)
- Centrifuge (Thermo Fisher Scientific, SorvallTM, model: RC-5B , catalog number: 8327-30-1004)
- pH meter (Fisher Scientific, catalog number: 13-644-928)
Manufacturer: Thermo Fisher Scientific, catalog number: 1112106 .
Procedure
Category
Microbiology > Microbial cell biology > Cell isolation and culture
Cell Biology > Cell isolation and culture > Transformation
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