Aug 11, 2022
Transforming E. coli
This protocol is a draft, published without a DOI.
- 1University of Wisconsin - Stout
Protocol Citation: Brian Teague 2022. Transforming E. coli. protocols.io https://protocols.io/view/transforming-e-coli-ce4ytgxw
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: August 10, 2022
Last Modified: August 11, 2022
Protocol Integer ID: 68472
Keywords: E. coli, transformation, plasmid, cloning
Abstract
Transformation is the process of inducing chemically competent E. coli to take up a plasmid. We do this in order to use E. coli as a "DNA copier" -- the bacterium takes up the plasmid, then copies it as it copies its own genomic DNA. We can grow up as many E. coli as we like in a liquid culture -- each of which has 20-50 copies of the plasmid -- then extract the plasmid DNA back out of the bacteria in the culture.
Guidelines
Time and temperature are critical parameters here -- make sure to follow the instructions very closely. For example, if the instructions read "heat shock in a water bath at 42 °C for exactly 00:00:30 ", don't grab your tube out of the ice bucket, wander over to the water bath, get distracted chatting to friends, etc. Instead, bring your ice bucket with your cells to the water bath, put the cells in the water bath for 30 seconds, and then put them immediately back on ice.
Materials
Equipment
- Water bath set to 42 °C
- Shaking incubator, set to 200 rpm, 37°C
- Ice bucket, with ice
Materials
- Ligation to transform
- Positive control plasmid
- SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S (or homemade SOC)
Note
Swirl the tube of SOC and check to make sure it's clear and NOT CLOUDY. It gets contaminated very easily!
- Glass beads 5 mmVWR ScientificCatalog #26396-596
- LB-Agar + kamamycin plates
- Competent E. coli (stored in the -80 °C freezer)
Note
Retrieve the two tubes of chemically competent E. coli cells from the -80 °C freezer and place them immediately on ice. Incubate the cells on ice 3-4 minutes to thaw.
Protocol materials
Glass beads 5 mmVWR InternationalCatalog #26396-596
Materials, Step 10
SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S
Materials, Step 7
Safety warnings
This protocol creates genetically modified organisms (GMOs) -- in particular, the E. coli bacteria become antibiotic-resistant. Make sure to dispose of contaminated plastics and cultures as instructed.
None of the materials in this lab are hazardous. HOWEVER, we are shedding nucleases -- enzymes that degrade DNA -- all the time. Wear lab coats and gloves to keep your samples nuclease-free.
Transformation
Transformation
32m 30s
32m 30s
Retrieve the two tubes of chemically competent E. coli cells from the -80 °C freezer and place them immediately on ice. Incubate the cells on ice 3-4 minutes to thaw.
Add 1 µL of DNA from your ligation to a tube of cells. Immediately after adding the DNA to the tube, flick several times to mix, then immediately return to ice.
Add1 µL of DNA from the transformation control to a separate tube of cells. Immediately after adding the DNA to the tube, flick several times to mix, then immediately return to ice.
Incubate the cells on ice for 00:30:00
30m
Heat shock the cells for exactly 00:00:30 in the 42 °C water bath.
Note
Bring your transformations ON ICE to the hot water bath. Move the cells from the ice bucket to the water bath, then BACK to the ice bucket.
30s
Incubate the cells on ice for 00:02:00
2m
Add 250 µL SOC Outgrowth Medium - 100 mlVWR InternationalCatalog #B9020S to each tube.
Tape each tube to the platform of an incubating shaker and shake 200 rpm, 37°C, 01:00:00
Plating
Plating
2m
2m
Label the kanamycin selection plates appropriately.
Note
There will be a LOT of plates generated by the class -- make sure you can tell which ones are yours and what is on each of them!
Note
I suggest you label the plates on the bottom, not on the lid.
Shake ~10 Glass beads 5 mmVWR InternationalCatalog #26396-596 onto each plate.
For each transformation, pipette 100 µL onto the center of the plate.
Cover the plates and shake the beads around to spread the cells out.
Dispose of the beads by tapping them into the waste container.
Incubate the plates upside down in an incubator, 37 °C Overnight
Note
Don't incubate for more than 18-24 hours!
2m