Oct 12, 2020
Protocol 2: CRISPR Transformation
Forked from Protocol 2: CRISPR Transformation
This protocol is a draft, published without a DOI.
- 1UCSC
- UCSC BME 22L
Protocol Citation: Ikenna Anigbogu 2020. Protocol 2: CRISPR Transformation. protocols.io https://protocols.io/view/protocol-2-crispr-transformation-bndama2e
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: October 12, 2020
Last Modified: October 12, 2020
Protocol Integer ID: 43138
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Materials
Perishables
55μL of 100ng/μL - Cas9 Plasmid Kanr
55μL of 100ng/μL - gRNA Plasmid Ampr
55μL of 1mM- Template DNA
1 - Sterile Water Tube
Non-perishables
1 - 1 mL Bacterial Transformation Buffer 25mM CaCl2, 10% PEG 8000
1 - Bag of Microcentrifuge Tubes
1 - Microcentrifuge Tube Rack
1 - 10-100 μL Variable Volume Adjustable Pipette
1 - Box 1-200 μL Pipette Tips
5 - Inoculation Loops
5 - 1.5mL Microcentrifuge Tubes containing LB Broth
BentoLab
Before start
The following video will demonstrate techniques used in E. Coli transformation. Keep in mind this is not the exact same protocol, but a general guide showing good lab practice.
The following video shows the necessity of making competent bacterials cells for transformation.
Mix Bacterial transformation solution with cultured E coli. Pipette 100uL of Bacterial Transformation mix into a microcentrifuge tube. Use an inoculation loop to lightly scrape E coli and transfer to the microcentrifuge tube. Use pipetting technique of aspirating and dispensing to homogenize the mixture.
Add CRISPR reagents. Retrieve tubes of Cas9 Plasmid, gRNA Plasmid, and Template DNA. Add 55uL of sterile water to each. Don’t forget to change the pipette tip. Make sure they are mixed well. In this order, pipette 10 uL from Cas9 Plasmid, gRNA Plasmid, and Template DNA into a centrifuge tube. Add each to the bottom of the competent cell mixture tube and follow by flicking your wrist or tapping the tube to the table to mix after each reagent addition.
Place the mixture into a PCR tube.
Incubate the tube in the refrigerator for 30 minutes. While you are waiting for the prepare the following:
LB media. Add 1.5 mL of room temperature water to the 1.5mL microcentrifuge tubes containing LB broth. Flick or tap the tube to mix. Once the mixture is completely dissolved, add 250 uL of the LB media into a new microcentrifuge tube.
On the bento lab, create a custom heating with PCR option. Make a setting where the PCR rack heats up to 42℃ for 30 seconds.
Take the tube out of the refrigerator and place in the PCR rack for 30 seconds.
Place contents of the PCR tube into the microcentrifuge tube containing 250 uL of LB Broth.
Incubate for 6-12 hours. If room temperature is cool, let the tube incubate for a longer period of time. If room temperature is warm, let the tube incubate for a shorter period of time.