Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2012/11/29

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Transfection of K562

  • Cell Density if at 1 mil/mg
  • Target Concentration of 500,000 cells/mL per well (6 well plate)
  • We need about 11uL (2000ng DNA/DNA sample in wells 1, 3, and 5)

Part 1

  1. Pellet cells to get rid of PS, aspirate (re-suspend 10mL into 15mL conical vial in PS free, spin for 3 min)
  2. Dilution (1/2, 1/4, 1/8) - make sure to pipet up and down
  3. Incubate while setting up transfection rxn

Part 2

  1. Transfer 4mL of OPTI-MEM to a 15mL conical vial (write on opti-mem when it was opened and initials; label opti-mem tube; make sure opti-mem, lipofectamine, and PLUS Reagent are at room temp)
  2. Add 570uL OPTI-MEM to sames and flick tube/invert
  3. Add 2.5uL PLUS Reagent to samples, incubate 5 min @ room temp
  4. Add 7.5uL of lipofectamine to the amples, incubate @ room temp for 30 min
  5. Add solution drop by drop ti each well
  6. Incubate (label wells w/ #'s and +/- DNA)

Wells

  1. 5 x 10^5 cells DNA (11uL DNA, 9uL H2O)
  2. 5 x 10^5 cells NEG (20uL H2O)
  3. 2.5 x 10^5 cells DNA (11uL DNA, 9uL H2O)
  4. 2.5 x 10^5 cells NEG (20uL H2O)
  5. 1.25 x 10^5 cells DNA (11uL DNA, 9uL H2O)
  6. 1.25 x 10^5 cells NEG (20uL H2O)


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