Mar 19, 2018
Version 2
E. coli Heat Shock Transformation V.2
- 1University of California, Riverside
Protocol Citation: Alex Rajewski 2018. E. coli Heat Shock Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.nwgdfbw
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 19, 2018
Last Modified: October 28, 2020
Protocol Integer ID: 10920
Abstract
This describes a method to transform a plasmid into homemade DH5α cells.
Materials
MATERIALS
Dry Block HeaterPromegaCatalog #Z3321
DH5α Competent Cells
SOC Media
Safety warnings
Competent cells and any materials that touch them should be disposed of in the biohazardous waste container.
Set up
Set up
Thaw competent cells on ice. This takes about 10 minutes. Also thaw the plasmid to be transformed.
Warm SOC media to room temperature.
250 µL SOC media per reaction
Note
LB media can also be used. Anecdotally, we haven't seen a difference between LB and SOC.
Heat a dry block to 42ºC, a shaker-incubator to 37ºC, and a cabinet-style incubator to 37ºC.
Label a 1.5mL eppendorf tube for each transformation reaction with the construct name and place it on ice. Label an agar plate with the construct, date, your initials, and plate media type (with antibiotic, if applicable).
Expected result
Plate: pYPQ131A 13 March 2018 AR LB+Tet
Heat Shock
Heat Shock
Add plasmid to the labeled eppendorf tube on ice.
2 µL plasmid
Add thawed competent cells to the tube with the plasmid and gently swirl with the pipet tip to mix.
45 µL competent cells
Note
DO NOT vortex. Competent cells have very specific and fragile cell membranes that can be damaged by vortexing.
Incubate the mixture on ice for 30 minutes.
00:30:00 Ice
Note
This time is fungible. Longer is better, but as short as 5 minutes will also work.
Place the mixture in the dry block at 42ºC for 30 seconds
00:00:30 Dry Block
Incubate the mixture on ice for 2 minutes.
00:02:00 Ice
Grow Up
Grow Up
Add SOC to the mixture and shake in an incubator
01:00:00 Shake at 37º & 250 rpm
250 µL SOC per reaction
(Optional) Spin the bacterial broth for 30 seconds at 6000 rpm, removed 180µL of supernatant, and gently resuspend the pellet in the remaining media to concentrate.
00:00:30 spin at 6000 rpm
Plate
Plate
Immerse a plate spreader in 70% ethanol to sterilize and then burn off the excess alcohol.
Pipet at least 100µL of the broth onto the labeled agar plate and spread it evenly across the surface of the agar with the sterile spreader. Restilerize the spreader.
Note
A larger volume can be used, or you can make two plates with different volumes (e.g. 100 and 50µL) to prevent getting colonies that are too close together.
Place the prepared plate (lid-down) into a 37ºC incubator overnight (16 hours)
Note
The plates can also be incubated at room temperature for several days (the weekend).