Oct 14, 2020
Transformation
This protocol is a draft, published without a DOI.
- 1Chung Shan Medical University
Protocol Citation: Huan Jui Chang 2020. Transformation . protocols.io https://protocols.io/view/transformation-bg4ajyse
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: June 03, 2020
Last Modified: October 14, 2020
Protocol Integer ID: 37730
Abstract
This protocol is designed to transform plasmids from distribution kits of iGEM 2017, 2018, 2019 as well as those synthesized by Mission Biotech with yT&A backbone into E.coli DH5-α competent cells. Chloramphenicol is used for selection in transformation when the plasmids are retrieved from iGEM distribution kits, while ampicillins is utilized when the plasmid are derived from Mission Biotech.
Attachments
Transformation.pdf
304KB
Guidelines
The ratio between competent cells and plasmid is 10 : 1. We will apportion 20 μL of competent cells for this protocol.
Safety warnings
Make sure that all the steps should be operated in laminar flow (except heat shock).
Before start
Follow the "preparation" section of protocol.
Preparation
Preparation
Sterilize styrofoam box with alcohol.
Turn on the UV light of the laminar flow for 00:10:00 to 00:15:00 in order to sterilize it.
Add ice into the styrofoam box and sterilize it again.
Place the styrofoam into the laminar flow.
Set the dry bath incubator at 42 °C
Protocol
Protocol
Apportion 20uL of E-coli DH5α into an eppendorf.
Safety information
From step 6 to 8, perform them on ice and in the laminar flow.
Add 2uL of target plasmid into the eppendorf and vortex
Ice the eppendorf for 3-5 min.
Protocol
Protocol
Heat shock the eppendorf for 1 min at 42°C in a dry bath incubator.
Ice the eppendorf for 3-5 min.
Add 100uL of antibiotic-free LB media into the eppendorf.
Incubate the eppendorf in 37 °C , shaking for 01:00:00
Spread the transformed bacteria on agar plate containing antibiotics.
Protocol
Protocol
Incubate the agar plate for Overnight (16-18 hours)