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August 10, 2003
 
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Protocol for Electroporation [back]  
  • Store Single Safe and Chamber in refrigerator (pre-chill) for 2 hours.
  • Prepare 2 new sterile Eppendorf tubes for each sample: one for mixing of DNA and E. coli cells and one for incubation after electroporation.
  • Put Eppendorf tubes used for mixing of DNA and competent cells on wet ice.
  • Add 0.5 ml of shotgun library DNA to the bottom of the tube carefully.
  • (Optional) Add 1 ml of control plasmid DNA purchased from Liftech BRL to the bottom of the tube carefully.
  • Thaw the competent cells on the wet ice.
  • Set the Single Safe on ice.
  • Aliquot 20 ml of competent cells for electroporation (ElectroMax DH10B) after gently pipetting to mix the cells.
  • Mix 20 ml of competent cells with DNA solution by gently by pipetting.
  • Add the cell-DNA mixture between the electrodes of the chamber in the Single Safe carefully, avoiding bubbles.
  • Close the cap of Single Safe tightly.
  • Charge and Trigger the pulse.
  • Add 980 ml of SOC medium immediately.
  • Put this culture medium containing E. coli cells into a new Eppendorf tube.
  • Incubate this tube into a 37°C water bath for 45 minutes (without shaking).
  • Add 100 ml of LB medium on the LB plate containing Ap, IPTG, and X-Gal.
  • Use 5 ml of cultured sample and spread on LB-ApR plate.
  • Add 150 ml of Glycerol to each tube and gently mix by pipetting.
  • Store remaining cells at -80°C till re-plating.
  • Incubate plates at 37°C overnight.

IMPORTANT: Ratio of white colonies to blue colonies should be 1:1 in an efficient and good shotgun library.

Prepare 96 plasmid DNA and check the length of each DNA. The shotgun library should have over 90% of total clones from the white colonies containing the correct size of insert.


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