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Pre-warm complete medium (DMEM with 10% FCS and 1% Pen Strep) in a 37◦ C water bath for 15–30 min. The pre-warmed complete medium is used in all subsequent steps. 2. Take out a frozen vial of HEK293T cells from a liquid nitrogen tank (typically contains 2–4×106 cells), while holding it, immediately place the vial in 37◦ C water, and gently swirl it until the liquid inside of the vial is completely thawed. 3. Take the vial out from water and spray it thoroughly with 70% ethanol to sterilize the surface, and place it in a laminar flow hood.
Primer design is crucial to the success of Gateway cloning and briefly the following procedure is undertaken. 1. Design the forward primers with a CACC overhang in front of the ATG start site to facilitate insertion of the gene into the entry vector pENTR/D-TOPO and keep 18 bp after the start site. 2. Design the reverse primers with 18 bp before the stop codon and remove the stop codon to ensure C-terminal fusion expression of GFP. 3. Check the reverse to ensure that there is no CACC at the 3 end; otherwise the correct orientation will not be maintained.
Add 1 ml of freezing medium to the cells and transfer to a freezing ampoule on ice. 9. Place the ampoules in a freezing container and leave at –70◦ C for 3 days, then store in liquid nitrogen until required. 10. Defrost cells by hand and tip into 30 ml culture medium in a T75 flask. See Note 4. 6. Printing Cell-Based Microarrays, Cell Addition to the Arrays and Transfection See Fig. 1 for array layout. Using the printing conditions below, the spot size on the microarrys is 140 μm with a 30-μm gap between each spot and a 170-μm gap between each sub-grid.