Nov 23, 2020
Recombineering
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. Recombineering. protocols.io https://protocols.io/view/recombineering-bpzhmp36
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 23, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44809
Disclaimer
DISCLAIMER: THIS IS A WORK IN PROGRESS. IT IS 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only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer-reviewed and may not have undergone a formal approval of any kind.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.
Before start
Template: if you are using pKD4 (kan cassette), you can use 200 ng of DNA for transformation. Plate on Kan 20 ug/mL plates (lower than normal). Pick to Kan 30 plates to verify really resistance.
Note
Dr. Fozo has not had an issue obtaining transformants on Kan 30 initially: only problematic if a weak RBS is driving Kan.
Be careful not to overgrow cells: we have seen a decrease in efficiency if the cells start approaching an OD 600 of 0.7 before you induce at 42.
If you are not using Kan 20 plates, use more DNA, and rescue for 2 hours.
Storing transformation mix: some people find that leaving the mix on the bench overnight increases the number of positive transformants when they plate out the next day.
Template
Template
Prepare a PCR product. Typically, 200-600 ng/transformation. See note below. If using a plasmid template, may have to digest using DpnI or another enzyme to remove the additional plasmid
Following purification, combine PCR products and ethanol precipitate. Resuspend products in water
Note
I usually combine 2-4 PCR products into 5 ul and use 2.5 ul for a first recombineering attempt.
Strains
Strains
Grow up 25 ml of a strain of choice at 30 or 32 degrees (do NOT use DY330. Use NM400, NM1100, or some such derivative).
When OD600 approximately 0.4, transfer to 42 degrees shaking water bath for 15 – 20 minutes.
After 15-20 minutes, place the flask in an icy slurry and swirl. Keep in icy slurry for about 2 minutes.
Spin at 4 degrees, 10 minutes, 4150 rpm.
Wash cells with 1 ml cold 10% glycerol at 4 degrees, three times
Resuspend cells in 100 µL cold 10% glycerol.
Electroporate: use 50 µL of cells + DNA. Immediately after electroporation, add 950 ul of SOC. Shake at 30 degrees for 1 hour. Plate 1/10 onto one plate, and concentrate the remaining 900 ul and plate to a second plate.