X-tremeGENE™ HP DNA Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Do not aliquot X-tremeGENE HP DNA Transfection Reagent; store in the original glass vials.

Upstream tips
Do not aliquot X-tremeGENE HP DNA Transfection Reagent; store in the original glass vials.

Publication protocol

SCGB1D2-rescue Construct
Reverse transcription-PCR (RT-PCR) was used to
amplify the entire coding sequence of the SCGB1D2 cDNA. The resultant PCR product was subsequently inserted into a pCMV6-Entry vector (Origene Technologies, Rockville, MD) bearing a C-terminal myc-DDK-tag. The X-tremeGENE HP DNA Transfection Reagent (Roche, Penzberg, Germany) was used to transiently transfect target cells with the resultant SCGB1D2-rescue construct. After incubation for 48 h, the cell pellet was resuspended in lysis buffer containing 20 mM HEPES (pH 7.4), 100 mM KCl, 2 mM MgCl2 , 0.5% Triton X-100, protease inhibitor cocktail tablets (Roche), and phosphatase inhibitor cocktail (Nacalai, Kyoto, Japan). The bicinchoninic acid (BCA) assay was used to determine the protein concentration of each ly ate, and an aliquot of each lysate was then diluted with sample buffer (50 mM Tris, 2% sodium lauryl sulfate (SDS), 0.1% bromophenol blue, and 10% glycerol) to a final concentration of 1-2 µg/ µL and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using anti-SCGB1D2 (sc-48327; Santa Cruz Biotechnology) and anti-myc (sc-789; Santa
Cruz Biotechnology) antibodies.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for X-tremeGENE™ HP DNA Transfection Reagent below.

Download manufacturer protocol

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