User:Isis Trenchard/Notebook/BioE44 Stuff/2010/03/19: Difference between revisions

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==Entry title==
==Making electrocompetant cells protocol==
* Insert content here...
* Modified for general electrocompetant cell prep (not specifically for recombineering): PMID: 19180090
#Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
#Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60. Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
#Wash the cell pellet with 20 ml of ice-cold H2O once and then resuspend in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 20–30 s at 4C.
#Wash the cells two more times with 1 ml of ice cold H2O. Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice.
#Mix 100 ng of BAC DNA with 50μl of electrocompetent cells and chill on ice for 5 min then transfer into a 0.1-cm cuvette. Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance). Also do a control electroporation where no DNA is added. After electroporation, immediately add 1 ml of SOC. Incubate cells at 37C for 1 h. Spin down cells for 20–30 s in a microcentrifuge. Resuspend the pellets in 200 ml of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow overnight.
 




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Making electrocompetant cells protocol

  • Modified for general electrocompetant cell prep (not specifically for recombineering): PMID: 19180090
  1. Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
  2. Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60. Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
  3. Wash the cell pellet with 20 ml of ice-cold H2O once and then resuspend in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 20–30 s at 4C.
  4. Wash the cells two more times with 1 ml of ice cold H2O. Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice.
  5. Mix 100 ng of BAC DNA with 50μl of electrocompetent cells and chill on ice for 5 min then transfer into a 0.1-cm cuvette. Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance). Also do a control electroporation where no DNA is added. After electroporation, immediately add 1 ml of SOC. Incubate cells at 37C for 1 h. Spin down cells for 20–30 s in a microcentrifuge. Resuspend the pellets in 200 ml of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow overnight.



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