Richard Lab:Electroporation of E. coli

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(New page: Back to Protocols 300px <center> '''This protocol is for the typical electro-transformation of ''E. coli'' done...)
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===Procedure===
===Procedure===
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1. Chill the # electroporation cuvettes by floating them in an ice bath.  
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# Chill electroporation cuvettes and DNA samples on ice.  
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2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.   
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# Remove # vials containing 100μl electro-competent cells from the -80°C freezer.  Thaw the vials on iceBe sure to include 2 vials for a negative and an unligated vector control.
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3. Prepare # micro-centrifuge tubes containing 900μl SOB media.
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# Prepare # micro-centrifuge tubes containing 900μl SOB media.
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4. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes)  
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# Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes)  
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5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.  
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# Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.  
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6. Place cells back on ice to ensure they remain cold.  
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# Place cells back on ice to ensure they remain cold.  
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7. Pippette 100μL of cell-DNA mixture to cuvette.   
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# Pippette 100μL of cell-DNA mixture to cuvette.   
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8. Wipe off excess moisture from outside of cuvette.  
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# Wipe off excess moisture from outside of cuvette.  
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9. Place cuvette in chamber of electroporator.  
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# Place cuvette in chamber of electroporator.  
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10. Pulse the cells by pressing button on electroporator twice.  
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# Pulse the cells by pressing button on electroporator twice.  
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11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.  
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# Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.  
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12. Let cells recover at room temperature for 30-60 minutes.  
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# Let cells recover at room temperature for 30-60 minutes.  
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13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.  
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# Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.  
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14. Leave remaining SOB-cell mixture on the bench-top overnight.  
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# Leave remaining SOB-cell mixture on the bench-top overnight.  
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15. If you don't have any transformants in the morning then plate the rest of the transformation.
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# If you don't have any transformants in the morning then plate the rest of the transformation.
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===Notes===
===Notes===
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* Our "1X TBE" is technically 0.5X TBE (5.3g/L Tris Base, 2.75g/L Boric Acid, and 2.9g/L EDTA) but for our purposes we call it 1X TBE.
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* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).
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** Correspondingly, the 10X TBE is officially 5X.
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Other protocols (Specificially the [[Knight Protocol]] were used extensively in the development of this protocol.  
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*Using TBE allows us to have such a high (150V) voltage.  If you use TAE you need to use a significantly lower voltage and your run time will be longer.  Despite that, TAE is advantagous in some cases, but [[User:Michael_A. Speer | Mike]] feels that TBE is better suited for his applications.  This is discussed in detail in the [[agarose gel electrophoresis | consensus  protocol]].  
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* Use the following table to determine the amount of agarose you want to use.
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[[Category:Protocol]] [[Category:In vivo]] [[Category:DNA]]
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<center>
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{|  border="1" cellpadding="5" cellspacing="0" align="center"
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|+ '''Choosing an Agarose Concentration'''
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! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)
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|-=
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| align="center"|0.5 || align="center"|1 - 30
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|-
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| align="center"|0.7 || align="center"|0.8 - 12
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|-
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| align="center"|1.0 || align="center"|0.5 - 10
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|-
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| align="center"|1.2 || align="center"|0.4 - 7
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|-
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| align="center"|1.5 || align="center"|0.2 - 3
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|}
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</center>
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[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]
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Back to [[Richard_Lab | Richard Lab]]
Back to [[Richard_Lab | Richard Lab]]

Revision as of 14:40, 23 June 2009

Back to Protocols

This protocol is for the typical electro-transformation of E. coli done in the Richard Lab.

The consensus electroporation protocol should be consulted if deviating from the procedure outlined here.

Procedure

1. Chill the # electroporation cuvettes by floating them in an ice bath. 2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes. 3. Prepare # micro-centrifuge tubes containing 900μl SOB media. 4. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) 5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells. 6. Place cells back on ice to ensure they remain cold. 7. Pippette 100μL of cell-DNA mixture to cuvette. 8. Wipe off excess moisture from outside of cuvette. 9. Place cuvette in chamber of electroporator. 10. Pulse the cells by pressing button on electroporator twice. 11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB. 12. Let cells recover at room temperature for 30-60 minutes. 13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C. 14. Leave remaining SOB-cell mixture on the bench-top overnight. 15. If you don't have any transformants in the morning then plate the rest of the transformation.

Notes

  • When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).

Other protocols (Specificially the Knight Protocol were used extensively in the development of this protocol.

Back to Richard Lab

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