Mar 12, 2026
E. Coli Bacterial Transformation
This protocol is a draft, published without a DOI.
- Tamra Lahom1
- 1Carleton College
Protocol Citation: Tamra Lahom 2026. E. Coli Bacterial Transformation . protocols.io https://protocols.io/view/e-coli-bacterial-transformation-jv2fcn8bp
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 12, 2026
Last Modified: March 12, 2026
Protocol Integer ID: 313127
Keywords: transformation of bacteria, replicating plasmid, bacterial origin of replication, plasmid, antibiotic resistance gene for use, transformation, bacterial origin, foreign dna, bacteria, antibiotic resistance gene, selectable marker in bacteria, studies in bacteria, antibiotic, replication, mammalian cell expression
Abstract
Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria.
Troubleshooting
Preparation
30m 45s
Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins)
Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up in 37°C incubator.
Heat Shock
30m 45s
Mix 2 µL of DNA (usually 10 pg - 100 ng) into 50 µL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
Heat Shock
30m 45s
Incubate the competent cell/DNA mixture on ice for 00:30:00
30m
Heat shock each transformation tube by placing the bottom of the tube into a 42°C water bath for00:00:45
Note
45 secs is usually ideal for E. coli however 30 - 60 is the range
Put the tubes back on ice for 00:05:00
Incubate Cells
1h
Add 500 μl LB or SOC media (without antibiotic) to the bacteria
Note
Make sure the media has no antibiotics.
Grow while shaking in 37°C shaking incubator for 1-2 hrs. 01:00:00
Note
This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate.
1h
Plating Cells
Plate some 150 µL and 300 µL of the transformation onto two LB agar plates containing the appropriate antibiotic.
Note
Plate under flames to maintain sterility
Incubate plates at 37°C overnight.