Team:Amsterdam/Notebook/Protocols/Transforming electrocompetent Cells

From 2011.igem.org

(Difference between revisions)
(→Transforming electrocompetent cells)
(→Procedure)
 
(3 intermediate revisions not shown)
Line 3: Line 3:
===Overview===
===Overview===
-
We use a protocol commonly used in our host lab provided by [https://2011.igem.org/Team:Amsterdam/Team/Advisors Lisette Anink]. The main advantage of electrocompetent cells compared to chemically competent cells is a higher level of competence (1-2 log higher). The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment.
+
We use a protocol commonly used in our host lab provided by [https://2011.igem.org/Team:Amsterdam/Team/Advisors Lisette Anink]. The main advantage of electrocompetent cells compared to chemically competent cells is a 1-2 orders of magnitude higher level of competence. The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment.
-
===Materials===
+
 +
===Materials===
 +
*Prewarmed [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Preparing_Media SOC]
 +
*[https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Making_electrocompetent_Cells Electrocompetent cells]
 +
*prechilled Sterilised elecroporation cuvettes
 +
**immersing cuvettes in 70% Ethanol and letting them dry works well
 +
** If you are in a hurry it is possible to dry cuvettes using pressurized air. This is less than completely sterile but it still works
 +
*Roundbottom aerated tubes
===Procedure===
===Procedure===
 +
*it is important to work on ice or in a cold room for the entire protocol
 +
*Thaw Competent cells on ice
 +
*Add 2 µl of ligation mix to slit corner of cuvette
 +
*Pipet competent cells on top of ligation mix
 +
*Incubate on ice for 30 minutes
 +
**it is possible to ignore this step but it is still recommended as it reduces transformation efficiency by a factor 2-5
 +
*Electroshock at 1250 volt. Monitor time
 +
**electroporation time should be higher than ~4.5. If the time end up at ~0.5 it usually means your cuvette broke due to too high salt concentration. It is, however, still recommended to plate the cells anyway.
 +
*add 900 µl of prewarmed SOC, pipette up and down and transfer to roundbottom aerated tube
 +
*shake for 90 minutes at 37°C
 +
*transfer to 1.5ml eppendorf tube
 +
*spin down at 5Krpm for 5 minutes
 +
*decant supernatant
 +
*resuspend pellet in last drop of SOC and plate on selective LB-agar plates using a plate spreader
 +
**The final downspin step may be skipped. Just spread the full 900 µl onto your plate
 +
**note that this will mean you will have to dry your plates for 1-2 hours prior to transferring to 37°C stove and it will leave an ugly texture on your plates. It does, however, ensure that all cells are transferred to your plate and eliminates some additional stress to your cells.
 +
*incubate plates overnight at 37°C
 +
 +
 +
 +
{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Latest revision as of 10:36, 21 September 2011

Contents

Transforming electrocompetent cells

Overview

We use a protocol commonly used in our host lab provided by Lisette Anink. The main advantage of electrocompetent cells compared to chemically competent cells is a 1-2 orders of magnitude higher level of competence. The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment.

Materials

  • Prewarmed SOC
  • Electrocompetent cells
  • prechilled Sterilised elecroporation cuvettes
    • immersing cuvettes in 70% Ethanol and letting them dry works well
    • If you are in a hurry it is possible to dry cuvettes using pressurized air. This is less than completely sterile but it still works
  • Roundbottom aerated tubes

Procedure

  • it is important to work on ice or in a cold room for the entire protocol
  • Thaw Competent cells on ice
  • Add 2 µl of ligation mix to slit corner of cuvette
  • Pipet competent cells on top of ligation mix
  • Incubate on ice for 30 minutes
    • it is possible to ignore this step but it is still recommended as it reduces transformation efficiency by a factor 2-5
  • Electroshock at 1250 volt. Monitor time
    • electroporation time should be higher than ~4.5. If the time end up at ~0.5 it usually means your cuvette broke due to too high salt concentration. It is, however, still recommended to plate the cells anyway.
  • add 900 µl of prewarmed SOC, pipette up and down and transfer to roundbottom aerated tube
  • shake for 90 minutes at 37°C
  • transfer to 1.5ml eppendorf tube
  • spin down at 5Krpm for 5 minutes
  • decant supernatant
  • resuspend pellet in last drop of SOC and plate on selective LB-agar plates using a plate spreader
    • The final downspin step may be skipped. Just spread the full 900 µl onto your plate
    • note that this will mean you will have to dry your plates for 1-2 hours prior to transferring to 37°C stove and it will leave an ugly texture on your plates. It does, however, ensure that all cells are transferred to your plate and eliminates some additional stress to your cells.
  • incubate plates overnight at 37°C



Retrieved from "http://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Transforming_electrocompetent_Cells"