Jan 07, 2025

Public workspaceBacterial Transformation Protocol

 Forked from Bacterial Transformation Protocol
This protocol is a draft, published without a DOI.
  • 1Iowa State University
Scott Nelson
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Protocol Citation: Scott Nelson 2025. Bacterial Transformation Protocol . protocols.io https://protocols.io/view/bacterial-transformation-protocol-dxay7ifw
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: January 07, 2025
Last Modified: January 07, 2025
Protocol Integer ID: 117816
Keywords: Bacteria, molecular biology
Abstract
Transformation of bacteria to for overexpression of protein, amplification of plasmid DNA, or other molecular biology techniques.
Materials
MATERIALS
ReagentLB agar plates with the proper antibiotic (e.g. Kanamycin)

ReagentLuria Broth Base (Miller's LB Broth Base)™, powderThermo FisherCatalog #12795027

ReagentHomemade chemically competent cellsNA

Before start
  • Prepare Luria broth (LB) agar plates and allow to set.
  • If pre-poured plates are being used, ensure the plates are warmed to 37° C.
  • Depending on the antibiotic marker present in the plasmid DNA, incorporate appropriate antibiotic in the LB agar.
  • Heat the water bath or heat block to 42° C.
  • Warm the sterile LB medium to room temperature (or 20-25° C in water bath).
  • Transfer competent cells from -80°C freezer to ice for 5-10 min or until thawed.
Add 10 ng to 200 ng of DNA directly to cells. This should be 0.5 to 2 µL. Incubate for 30 min on ice.
  • Heat shock cells for 45 seconds at 42°C in a heat block. (Do not go over 45 seconds!) You can kill bacteria by keeping them at high temps for too long.
Transfer cells to ice and incubate for 5 minutes.
Add 450 µL of sterile LB or SOC media.
  • Incubate tube for 1 h at 37°C.
Add 100 µl of cells after incubation to warmed LB plate and spread cells.
  • Incubate plates overnight at 37°C with the plate facing down.