TITLE: Over-Agar Antibiotic Plating
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for over-agar antibiotic plating. To see the full abstract and additional resources, visit </span><a href="https://www.addgene.org/protocols/over-agar-antibiotic-plating" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/over-agar-antibiotic-plating/</span></a></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data: Selection of E.coli on LB-agar using different concentrations of carbenicillin plated over-agar.</span></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh6.googleusercontent.com/bEqeXitoKK7jJOKWKU-ZF_jdJZMvepxXv6lbpn0W5uAmNUor2yoN6cP8HVHZCgPJwgdhi0KIgWDp91XUVHjZfCrSCFWgZlaDi7HDk3ZDtACbaIodauO_rZayJrn8ZcPmsz2m_i1o" /></div><div class = "text-block"><span style = "font-weight:bold;">Control Plate with No Carbenicillin</span></div><div class = "text-block"><span>Plate shows a lawn of </span><span style = "font-style:italic;">E. coli</span><span> and no selection.</span></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh5.googleusercontent.com/612pOiuLcda6-aixJjwp24OzXWwYMg63B2uM6IYR2QKiehujCnLfTd7xesEY2OxeNokJuSMIyjlBC8MK6x4HMw3Z5opWDOMm1Exrb8G4F-IZIsApLXuEV8QJDc-EWN4EN-RPiL-R" /></div><div class = "text-block"><span style = "font-weight:bold;">150 µL of 0.1 mg/mL Carbenicillin plated over-agar</span><span style = "font-weight:bold;"></span></div><div class = "text-block"><span>Plate shows a lawn of </span><span style = "font-style:italic;">E. coli</span><span> and no apparent selection.</span></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh3.googleusercontent.com/1A4UQNdFMqJv_TNGlMBTtuENk5DEuZD1Uq_TDkCVbeSSu1FXCNVSJFQr4nJTxvDwXNqakfDnZSoxxehN6QPmsu38dlLQPTG_QNOEuVG-EQijkOIpbEqr3SA7MxTOwxiOcRDpkM3m" /></div><div class = "text-block"><span style = "font-weight:bold;">150 µL of 1 mg/mL Carbenicillin plated over-agar</span><span style = "font-weight:bold;"></span></div><div class = "text-block">Plate shows several individual colonies and effective selection.</div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh6.googleusercontent.com/rVCizz_3AA4FhT4PV38juXtsy2IHyQRC6XJu78rCHG0BM7JIjpYREXRKCZitR0S1yZN6JpzT7LVhuW8IQUHzo0mJzD1lK6JgtvrxdK6vxtwkVXl742K6AhmesIkrUu4lwndo37TQ" /></div><div class = "text-block"><span style = "font-weight:bold;">150 µL of 2 mg/mL Carbenicillin plated over-agar</span><span style = "font-weight:bold;"></span></div><div class = "text-block">Plate shows less individual colonies than the 1 mg/mL plate and effective selection.</div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh5.googleusercontent.com/rmvAf971G3zFQ1uujbwgz2FAwHgPprOewSVIJf_zr-BfDZvfyjfcYuLHCRJw1JITCOFxxEuJKgJUoPxkN0Z0igIH5YDmv4Wyu1YZvyL20K8M6fSn8IwWQIYRNxliu2BGW6SJ_CTe" /></div><div class = "text-block"><span style = "font-weight:bold;">150 µL of 4 mg/mL Carbenicillin plated over-agar</span><span style = "font-weight:bold;"></span></div><div class = "text-block">Plate shows several individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection.</div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh5.googleusercontent.com/HTok4f_8izTbAciwbE9pMsI5ZjaMias4nPlk1WYqxh9HDq-z-ewbJPIoSpsxUytrO_6MhRlcfgOMIvtdIVjgIHCBzgAvIc-0vhsHTWybRo2ucbps_Tlh-zKa1Qj12zzONWQ5ZLMi" /></div><div class = "text-block"><span style = "font-weight:bold;">Selection Curve of Transformed E. coli after Over-Agar Plating of Carbenicillin.</span><span> The above graph displays the stock concentration of Carbenicillin stock used (150 µL per plate). Please note we have found that there is generally a broad range of antibiotic concentrations that will work for this assay, and the above result represents a single experiment. For publishable data, the experiment would need to be repeated to account for variability.</span></div></div>

[STEPS]
?. [Day 1]
Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium.
The concentration of antibiotic required for effective over-agar selection has been empirically determined. See selection curve below.
Carbenicillin is used here in place of ampicillin because carbenicillin is more stable, so it is potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). It is, however, more expensive.
?. [Day 1]
With a 6 cm diameter petri plate containing solidified LB-agar, pipette  of carbenicillin on top of the agar and gently spread over the surface until the liquid is mostly absorbed (there is a very small visible volume of pooled liquid remaining on the surface).
150 µl
We use the micropipette tip itself to do the spreading; the tip is gently bent to create an “L” shape, and then used like a cell spreader. Several other devices may be used for this purpose, provided that they fit your petri plate.
?. [Day 1]
Incubate the plate at  for at least  with the lid on to give the antibiotic time to more fully absorb.
0 Room temperature
?. [Day 1]
During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our transformation page for a detailed heatshock transformation protocol.
?. [Day 1]
Plate  of transformed E. coli/rescue media suspension onto the agar and gently spread over the surface until the liquid is mostly absorbed.
50 µl
The spreading of cells can be done in the same way as the antibiotic, using either a bent micropipette tip or other cell spreading device that fits the plate.
?. [Day 1]
Incubate plates at  for .
37 °C
?. [Day 2]
Observe plates for colony formation. Shown below are the results from an experiment optimizing the concentration of carbenicillin, plated over-agar for selection of transformed E. coli.