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
- Pellet cells to get rid of PS, aspirate (re-suspend 10mL into 15mL conical vial in PS free, spin for 3 min)
- Dilution (1/2, 1/4, 1/8) - make sure to pipet up and down
- Incubate while setting up transfection rxn
Part 2
- 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)
- Add 570uL OPTI-MEM to sames and flick tube/invert
- Add 2.5uL PLUS Reagent to samples, incubate 5 min @ room temp
- Add 7.5uL of lipofectamine to the amples, incubate @ room temp for 30 min
- Add solution drop by drop ti each well
- Incubate (label wells w/ #'s and +/- DNA)
Wells
- 5 x 10^5 cells DNA (11uL DNA, 9uL H2O)
- 5 x 10^5 cells NEG (20uL H2O)
- 2.5 x 10^5 cells DNA (11uL DNA, 9uL H2O)
- 2.5 x 10^5 cells NEG (20uL H2O)
- 1.25 x 10^5 cells DNA (11uL DNA, 9uL H2O)
- 1.25 x 10^5 cells NEG (20uL H2O)
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