Apr 29, 2022

Public workspaceOptimizing the electroporation parameters for Heterometopus palaeformis (strain RAJCA)

DOI
dx.doi.org/10.17504/protocols.io.rm7vzy78rlx1/v1
  • 1Harvard University;
  • 2University of Rhode Island;
  • 3Woods Hole Oceanographic Institution
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzy78rlx1/v1
Protocol Citation: Fatma Gomaa, Johana Rotterova, Roxanne Beinart, Maria Pachiadaki, Virginia Edgcomb 2022. Optimizing the electroporation parameters for Heterometopus palaeformis (strain RAJCA). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy78rlx1/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: April 29, 2022
Last Modified: April 29, 2022
Protocol Integer ID: 61698
Abstract
Cell viability after incubation with different electroporation buffers,
Phosphate buffered saline (PBS) is used as an electroporation buffer for many eukaryotic cells. However, we observed that the PBS has a detrimental effect on the cell, causing disruption in H. palaeformis cell membrane. We then tested the tolerance of H. palaeformis to three different electroporation buffers:
A-Gene Pulser Electroporation Buffer(BIO-RAD, USA)
B-Iso-osmolar Buffer (Eppendorf, USA)
C-Hypo-osmolar Buffer (Eppendorf, USA)
Cells were incubated in each buffer independently for 10 minutes and observed for their viability under inverted microscopy. For all the three tested buffers, we noticed that once buffer was added, the cells showed instant osmic shock, include loss of motility and relative shrinking in the cell membrane (Fig. 1). After 10 minutes incubation period in each buffer, cells were transferred to their Cerophyl culture medium to recover. We observed progressive recovery of the cells. Table 1 shows the time that took the cells to completely recovered and the percentages of the recovered cells after incubation at the three different electroporation buffers.

Table 1
Electroporation buffer Duration of recovery Percentage of the recovered cells
Gene Pulser 10 minutes >90%
Iso-osmolar 10 minutes 70%
Hypo-osmolar 2-5 minutes 70%



Fig.1. Light microscopy images showing the effect of the different electroporation buffer on the H. palaeformis cell membrane; PBS has detrimental effect on the cell causing disruption of cell membrane.