siRNA MXD3

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA1: 5′-AUG GAC UAA AAG GAC CCU Utt-3′; siRNA2 - 5′-AGG ACC CUU GUG UGG GAA Ctt-3′ siRNA concentration-100 pmol siRNA per well. Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 5 min at RT and add to cells. Incubate cells for 48 days at 37°C.

Protocol tips
siRNA1: 5′-AUG GAC UAA AAG GAC CCU Utt-3′; siRNA2 - 5′-AGG ACC CUU GUG UGG GAA Ctt-3′ siRNA concentration-100 pmol siRNA per well. Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 5 min at RT and add to cells. Incubate cells for 48 days at 37°C.

Publication protocol

DAOY cells were grown in MEM medium supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate and penicillin/streptomycin. For transient expression, cells were transfected at 90–95% confluence using FuGene HD (Roche) at a 5∶2 ratio (µl reagent:µg DNA) or Lipofectamine 2000 (Invitrogen) at a 3∶1 ratio, according to manufacturer’s instructions. Two µg of DNA were used per well in 6-well plates. siRNA transfections were carried out using 2.5 µl Lipofectamine-2000 (Invitrogen) and 100 pmol siRNA per well in 6-well plates with cells at 80–90% confluency.

For the generation of stable lines, cells were transfected as described above in 100 mm dishes. After 48 hrs, G418 (Cellgro) was added to a final concentration of 800 µg/ml and colonies were picked and expanded after 2–3 weeks. All stable cell lines used in the experiments described here were maintained in culture for more than 40 passages with no obvious change in phenotype.

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Manufacturer protocol

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