pSilencer 2.1-U6 Hygro

shRNA gene silencing Human - SHSY5Y Beclin 1

Experiment

shRNA gene silencing Human - SHSY5Y Beclin 1

Product

pSilencer 2.1-U6 Hygro from Thermo Fisher Scientific

Manufacturer

Thermo Fisher Scientific

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Protocol tips

Upstream tips	Protocol tips	Downstream tips
	SH-SY5Y, Dulbecco’s modified Eagle’s medium (DMEM, 12100-046; Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS, SH30088.03; Thermo Scientific HyClone), 100 U/ml penicillin, 100 µg/ ml streptomycin and 2 mM glutamine. Cells were maintained in a humidified 10% CO2 atmosphere at 37°C. A human Beclin 1 shRNA hairpin (target sequence: CTC AGG AGA GGA GCC ATT T) was cloned into the HindIII and BamH1 sites of pSilencer 2.1-U6 Hygro. Beclin 1 shRNA and control scrambled shRNA plasmids were transfected into SH-SY5Y cells and selected with 50 µg/ml Hygromycin.

Protocol tips
SH-SY5Y, Dulbecco’s modified Eagle’s medium (DMEM, 12100-046; Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS, SH30088.03; Thermo Scientific HyClone), 100 U/ml penicillin, 100 µg/ ml streptomycin and 2 mM glutamine. Cells were maintained in a humidified 10% CO2 atmosphere at 37°C. A human Beclin 1 shRNA hairpin (target sequence: CTC AGG AGA GGA GCC ATT T) was cloned into the HindIII and BamH1 sites of pSilencer 2.1-U6 Hygro. Beclin 1 shRNA and control scrambled shRNA plasmids were transfected into SH-SY5Y cells and selected with 50 µg/ml Hygromycin.

Protocol tips

SH-SY5Y, Dulbecco’s modified Eagle’s medium (DMEM, 12100-046; Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS, SH30088.03; Thermo Scientific HyClone), 100 U/ml penicillin, 100 µg/ ml streptomycin and 2 mM glutamine. Cells were maintained in a humidified 10% CO2 atmosphere at 37°C. A human Beclin 1 shRNA hairpin (target sequence: CTC AGG AGA GGA GCC ATT T) was cloned into the HindIII and BamH1 sites of pSilencer 2.1-U6 Hygro. Beclin 1 shRNA and control scrambled shRNA plasmids were transfected into SH-SY5Y cells and selected with 50 µg/ml Hygromycin.

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Discussion

5 years ago

Author: Italy

Is a knockdown using shRNA permanent?

Is a knockdown using shRNA permanent and if not is there a known duration?

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Papers

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Manufacturer protocol

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