Nov 23, 2020
Removal of FRT flanked antibiotic resistance gene
This protocol is a draft, published without a DOI.
- 1Department of Microbiology, University of Tennessee
Protocol Citation: Elizabeth Fozo 2020. Removal of FRT flanked antibiotic resistance gene. protocols.io https://protocols.io/view/removal-of-frt-flanked-antibiotic-resistance-gene-bpw8mphw
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 20, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44736
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Abstract
Protocol: Removal of antibiotic resistance marker with FRT sites using pCP20 plasmids
Protocol based on Datsenko 2000
Protocol based on Datsenko 2000
Transform with pCP20 following transformation protocol. Rescue at 30 °C and spread cells on Amp plates.
Incubate overnight at 30 °C .(While cells are growing at 30 °C the flippase should be in action and should flip out the resistance marker at FRT sites.
Optional additional step: Streak colonies on Amp, incubate at 30 °C overnight. Will allow for a longer period of flippase action. (This helps if perform protocol first time without it and get low efficiency of flipping out of antibiotic marker)
Patch multiple colonies (~25) onto LB only, LB + Ab with frt sites (eg.Cm or Kan), and LB+Amp and incubate at 42 °C overnight. (pCP20 is a temperature-sensitive plasmid and this will help lose the pCP plasmid.)
Take a colony and serially patch onto the plates, starting with LB only, in the order mentioned above. Incubate overnight at the appropriate temperature.
Original protocol says to patch on LB last to ensure adequate cells are transferred onto antibiotic plates, but BB suggests LB first.
Interpretation of Results
No growth on any antibiotic plate – successful removal of marker and nopCPin cells
Growth on Amp plates – pCP20 not lost from cells
- Perform second isolate streak and incubation at 42 °C overnight and repeat steps 3 to 4.
Growth on Ab (to be flipped out) plates – failed removal (see optional step 2a for possible troubleshooting)