Ellis:Back Door/Protocols/Tranformation

From OpenWetWare

< Ellis:Back Door(Difference between revisions)
Jump to: navigation, search
Current revision (05:55, 14 June 2011) (view source)
 
Line 1: Line 1:
=E. coli Transformations using Electroporation=
=E. coli Transformations using Electroporation=
 +
A negative control for your experiments should be transformed alongside your samples.
<ol>
<ol>
<li>Get required amount of electrocompetent cells from the -80°C freezer and let them thaw slowly on ice. Usually 1 tube (50μL / tube) per transformation.</li>
<li>Get required amount of electrocompetent cells from the -80°C freezer and let them thaw slowly on ice. Usually 1 tube (50μL / tube) per transformation.</li>
Line 6: Line 7:
<li>Add your competent cells to each transformation by targeting the drop of DNA previously deposited.</li>
<li>Add your competent cells to each transformation by targeting the drop of DNA previously deposited.</li>
<li>Keeping the cuvettes on ice, add 50μL of the competent cells and mix by pipetting up and down trying to avoid creating air bubbles</li>
<li>Keeping the cuvettes on ice, add 50μL of the competent cells and mix by pipetting up and down trying to avoid creating air bubbles</li>
-
<li></li>
+
<li>Take your ice bucket, rich media (2YT or SOC etc.), a P1000 pipette and according tips to the Electroporator machine</li>
-
<li></li>
+
<li>Set the electoporator to settings "Bacteria" and "ms"</li>
 +
<li>Thoroughly dry the cuvette external walls using paper to ensure a good connection</li>
 +
<li>Place the cuvette in the slideable holder with the cuvettes protuberance towards the electrodes (slide the extra bit of plastic in the separation between each half of the slider)</li>
 +
<li>Electroporate by pressing the lighting bolt button and record the "ms" value that appears. Ideally, the value should lie between 4 and 6.</li>
 +
<li>Quickly add 2-3mL of rich media to your cuvette, resuspend gently by pipetting up and down and let sit at room temperature until you are done with all the cuvettes</li>
 +
<li>Transfer the contents of the cuvette to a labeled eppendorf tube and incubate, shaking at 37°C for 30-45 minutes</li>
 +
<li>Plate the contents of the eppendorf onto appropriately selective media using a spreader and incubate at 37°C overnight</li>
 +
<li>The following day, check for colonies on the plates. You should often allow a minimum of 16 hours for the colonies to appear.</li>
</ol>
</ol>

Current revision

E. coli Transformations using Electroporation

A negative control for your experiments should be transformed alongside your samples.

  1. Get required amount of electrocompetent cells from the -80°C freezer and let them thaw slowly on ice. Usually 1 tube (50μL / tube) per transformation.
  2. Take the appropriate amount of electroporation cuvettes from the -20°C freezer (1 per transformation) and label them accordingly
  3. Place 1-2μL of the DNA you wish to transform against the side of the cuvette, between the electrodes.
  4. Add your competent cells to each transformation by targeting the drop of DNA previously deposited.
  5. Keeping the cuvettes on ice, add 50μL of the competent cells and mix by pipetting up and down trying to avoid creating air bubbles
  6. Take your ice bucket, rich media (2YT or SOC etc.), a P1000 pipette and according tips to the Electroporator machine
  7. Set the electoporator to settings "Bacteria" and "ms"
  8. Thoroughly dry the cuvette external walls using paper to ensure a good connection
  9. Place the cuvette in the slideable holder with the cuvettes protuberance towards the electrodes (slide the extra bit of plastic in the separation between each half of the slider)
  10. Electroporate by pressing the lighting bolt button and record the "ms" value that appears. Ideally, the value should lie between 4 and 6.
  11. Quickly add 2-3mL of rich media to your cuvette, resuspend gently by pipetting up and down and let sit at room temperature until you are done with all the cuvettes
  12. Transfer the contents of the cuvette to a labeled eppendorf tube and incubate, shaking at 37°C for 30-45 minutes
  13. Plate the contents of the eppendorf onto appropriately selective media using a spreader and incubate at 37°C overnight
  14. The following day, check for colonies on the plates. You should often allow a minimum of 16 hours for the colonies to appear.
Personal tools