ON-TARGETplus Human MET (4233) siRNA - SMARTpool

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration: 30 nM–300 nM. Post nucleofection,Incubate the cells in humidified 37°C/5% CO2 incubator until analysis. Knockdown can be detected after 6–8 hours.

Protocol tips
siRNA concentration: 30 nM–300 nM. Post nucleofection,Incubate the cells in humidified 37°C/5% CO2 incubator until analysis. Knockdown can be detected after 6–8 hours.

Publication protocol

Knockdown in H1299 and MDA MB231 was achieved by transfection of siRNA using Hiperfect (Qiagen) according to the protocol of the manufacturer, and constructs were expressed using Genejuice (Merck-Millipore) according to the protocol of the manufacturer. Knockdown and overexpression in HT29 and A431 cells were achieved by transfection of siRNA or plasmids using the AMAXA nucleofection method, solution V, and protocols X-001 and X-005, respectively.

The following siRNA oligos were used: control siRNA 1, 5′-GCAACGGCAUUCCACCUUU(TT)-3′; control siRNA 2 control pool of four siRNAs (Dharmacon, catalog no. D-001810-10−20); RCP (SMARTpool of four siRNAs (Dharmacon, catalog no. L-015968-00-0005); p53, 5′-GACUCCAGUGGUAAUCUAC(TT)-3′; p63 1, 5′-UGAACAGCAUGAACAAGCU(TT)-3′; p63 2, 5′-UGACUUCAACUUUGACAUG(TT)-3′; and MET (SMARTpool of four siRNAs (Dharmacon, catalog no. L-003156-00-0005); Dicer 1, 5′-GCU CGA AAU CUU ACG CAA A (TT)-3′; and Dicer 2, 5′-CCA CAC AUC UUC AAG ACU U (TT)-3′. All siRNA oligos were used as a combination of two or four siRNAs, and each individual siRNA was tested for efficiency of knockdown and off-target effects.

For siRNA rescue experiments, MDA MB231 and HT29 cells were transfected with the AMAXA nucleofection method, solution V, and protocols X-001 and X-005 using a combination of 2 μg of plasmid and 120 pmol of p53 siRNA for 8 × 106 cells.

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Manufacturer protocol

Download the product protocol from Dharmacon for ON-TARGETplus Human MET (4233) siRNA - SMARTpool below.

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