E. coli Transformation Protocol

Overview

This lecture describes the enhanced transformation protocol for introducing plasmid DNA into E. coli using calcium chloride, including step-by-step preparation, transformation, and visualization procedures.

Preparation of Source Plates

• Remove a single Backdo bead from the E. coli GFP host vial with a sterile loop and place it on the agar source plate edge.
• Add 10 µL of recovery broth to dissolve the bead and streak for colony isolation using primary and secondary streaking.
• Label, cover, and incubate plates inverted at 37°C for 18 to 22 hours.

Preparation of E. coli Starter Culture

• Use a source plate cultured for 18–22 hours and a 37°C water bath.
• Add 30 mL recovery broth to a 50 mL tube, label "E. coli culture."
• Collect bacteria with a sterile loop (match-head size), resuspend in broth, and vortex if available.
• Incubate at 37°C water bath for 60 minutes.
• Aliquot 1 mL culture into 1.5 mL microcentrifuge tubes, label, and place on ice.
• Store tubes on ice for up to 24 hours or pellet cells by centrifugation and store at 4°C.

Preparation of Competent Cells

• Centrifuge tubes to pellet cells, pour off/pipette out supernatant.
• Add 200 µL ice-cold calcium chloride, gently resuspend, and incubate on ice for 10 minutes.
• Centrifuge again, remove supernatant, add 100 µL ice-cold competent cell solution, and resuspend.
• Store on ice or in freezer for up to 48 hours.

Transformation Procedure

• Label one tube "+ DNA" (with plasmid) and the other "– DNA" (without plasmid).
• Add 150 µL ice-cold calcium chloride to each tube.
• Add 10 µL plasmid DNA to "+ DNA" tube.
• Incubate both tubes on ice for 10 minutes.
• Heat shock at 42°C for 45 seconds, then return to ice for 2 minutes.
• Add 250 µL recovery broth, mix, and incubate at 37°C for 10 minutes.
• Label plates and plate 250 µL from each tube onto appropriate plates.
• Spread cells evenly using an inoculating loop.

Visualization

• Incubate plates overnight at 37°C.
• Use blue light or UV light to visualize transformation results.

Key Terms & Definitions

• Competent cells — Bacterial cells treated to allow uptake of foreign DNA.
• Calcium chloride protocol — Method using CaCl₂ to increase cell membrane permeability for transformation.
• Plasmid DNA — Circular DNA molecule used for genetic transformation.
• Heat shock — Brief exposure to high temperature to facilitate DNA uptake.
• Recovery broth — Nutrient-rich solution aiding cell recovery post-transformation.

Action Items / Next Steps

• Visualize plates with UV or blue light after overnight incubation.
• Ensure proper labeling and storage of cultures and competent cells as instructed.