Oct 07, 2022

Public workspaceElectroporatic transformation of Rhodobacter sphaeroides

DOI
dx.doi.org/10.17504/protocols.io.eq2ly7d6wlx9/v1
  • 1Argonne National Lab
Jaya K Yakha
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly7d6wlx9/v1
Protocol Citation: Jaya K Yakha, Deborah K Hanson, Rosemarie Wilton, laible 2022. Electroporatic transformation of Rhodobacter sphaeroides. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7d6wlx9/v1
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: September 12, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 69895
Abstract
The expression host for R. sphaeroides DrshI wasconstructed by deleting Type II restriction enzyme RshI (locus tag RSP_3759; recognition sequence CGATCG) from strain DD11 is suitable for transformation by either chemical or electrical procedures.
Materials
- 15 ml sterile tubes
- 1 ml microcentrifuge tubes
- Sterile glass beads
- Competent cells (Drsh1)
- Ice
- Electroporation tube
- Incubator at 330C
- Media plate with appropriate Antibiotic selection
- GYCC liquid medium
- Centrifuge

In a microfuge tube on ice, mix Amount40 µL cells with 100 ng of plasmid DNA.


Turn on the electroporator and select the voltage to 2500 V with other setting of 25uF and 400 ohm.

Transfer the cells-DNA mixture to a chilledThikness2 mm electroporation cuvette on ice.

Transfer the cuvette to the pulse chamber and shock to desired number of pulses (only once) (if doing multiple pulses, do first pulse and return cuvette to ice for one minute before doing next (and additional) pulses. Duration00:01:00 e on ice between pulses.

1m
Return the cuvette to ice and add 1ml GYCC immediately; record the time constant and the voltage achieved for each pulse.

Transfer cells/GYCC from electroporation cuvette to a Falcon 2059 culture tube (15ml). Incubate at Temperature35 °C with slow shaking (Shaker150 rpm ) for a Duration04:00:00 outgrowth period.

4h
Pellet the cells in microcentrifuge tube and spread onto GYCC agar containing appropriate antibiotic. Incubate plates at Temperature33 °C for 2-4 days.