Team:University of Chicago/Notebook/Transformations

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(New page: ==Chemocompetent cells== ===General transformation== #Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) * This is at 10 pg/_l or 10-5 _g/_l * This can be made by ...)
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Revision as of 15:03, 8 August 2008

Chemocompetent cells

=General transformation

  1. Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
  • This is at 10 pg/_l or 10-5 _g/_l 
* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
  1. Hold on ice 0.5 hours
  2. Heat shock 60 sec at 42C
  3. Add 250 _l SOC
  4. Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
  • using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
  • For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol

and tetracycline resistant, we find growing for 2 hours yields many more colonies

  • Ampicillin and kanamycin appear to do fine with 1 hour growth
  1. Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
  • Good cells should yield around 100 - 400 colonies
  • Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
  • We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA
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