Jul 23, 2020
Bacterial Transformation Protocol
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
- George Lab @ UCSDTech. support email: olgeorge@ucsd.edu
Protocol Citation: Olivier George, Sierra Simpson 2020. Bacterial Transformation Protocol . protocols.io https://protocols.io/view/bacterial-transformation-protocol-82yhyfw
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: November 05, 2019
Last Modified: July 23, 2020
Protocol Integer ID: 29496
Keywords: Bacteria, molecular biology
Abstract
Transformation of bacteria to amplify DNA for cloning, virus production, or other molecular biology techniques.
Materials
MATERIALS
LB agar plates with the proper antibiotic (e.g. Kanamycin)
MACH1 or DH5a or TOP10 or NEB stable or etc for DNA purification/miniprep
Luria Broth Base (Miller's LB Broth Base)™, powderThermo FisherCatalog #12795027
14-956-9C Culture Tubes
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 (DH5a) from -80 to wet ice for 5-10 min or until thawed.
Add 1ng to 50ng of DNA directly to cells. Incubate for 10 minutes on ice.
Heat shock cells for 45 seconds at 42C 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 cells to sterile culture tubes with 5ml of LB + selection antibiotic. Ensure lid is not tight to ensure proper aeration of the cultures.
Incubate starter culture for 1h at 230rpm in a shaker at 37C.
Add 100ul of starter culture after incubation to warmed LB plate. Spread using roller beads.
Incubate plates o/n at 37C with the plate facing down to avoid desiccation of the agar.
In the morning, select colonies and culture in 3-5ml of Luria Broth (LB) overnight for Mini-prep in following morning.
Isolate the DNA from the culture.
Digest the plasmid with appropriate restriction enzyme and visualize with gel electrophoresis to determine if the plasmid insert is correct.
Send out the plasmid for sequencing before using to make virus, or functional studies.