water:	autoclaved double-deionized water or commercial ultrapure DNase/RNase-Free distilled water
2× annealing buffer:	20 mM Tris pH 7.8
	100 mM NaCl
	0.2 mM EDTA
1× TE buffer:	10 mM Tris pH 8.0
	1 mM EDTA
spectinomycin:	set up 10 mg/ml stock in water and filter sterilize, aliquot and store at −20°C
ampicillin:	set up 100 mg/ml stock in water and filter sterilize, aliquot and store at −20°C
LB medium:	25 g/l pre-mixed LB medium dissolve in double-deionized water and autoclave to sterilize
LB plates:	25 g/l LB medium and 15 g/l agar in double-deionized water
	- add a stir bar and autoclave to dissolve agar and sterilize the solution
	- cool down while spinning at low speed on a magnetic stir plate
	- once the solution has cooled down to touch, add sterile-filtered antibiotics, e.g. 50 μg/ml spectinomycin or 200 μg/ml ampicillin, before pouring plates
2M CaCl2:	dissolve in double-deionized water and filter-sterilize, store at room temperature
0.1× TE:	1 mM Tris pH 8.0
	0.1 mM EDTA
	dissolve in double-deionized water, filter-sterilize, aliquot and store at −20°C
2× HBS:	dissolve 32.8 g NaCl, 0.42 g Na2HPO4, and 23.8 g HEPES in double-deionized water
	- adjust pH to 6.98 and bring to final volume of 2 l
	- set 500 ml aside and adjust the pH of the remaining solution to 7.00
	- set 500 ml aside and adjust the pH of the remaining solution to 7.02
	- set 500 ml aside and adjust the pH of the remaining solution to 7.04
	- filter-sterilize all four solutions, aliquot and store at −20°C
	To identify the pH/solution with the highest transfection efficiency
	- plate 5 × 106 HEK-293-T cells each in four 10 cm dishes
	- the next day, prepare four 1.7 ml microfuge tubes each with
	660 μl 0.1× TE,
	90 μl 2 M CaCl2,
	20 μg targeting vector (EmGFP or mRFP)
	10 μg pMDLg/pRRE,
	6 μg pMD2.g, and
	5 μg pRSV-Rev
	- mix in one of the four HBS solution into each tube, vigorously pipet up and down to mix
	- incubate 20 min at room temperature
	- add transfection solutions to cells and return cells to incubator
	- the next day, choose the HBS solutions with best transfection efficiency based on the percentage of fluorescent cells in the culture, fluorescence intensity and cell viability.
	Note: cells and medium contain virus.
	We usually find that one or two of the four HBS solutions perform significantly better than the others and we test every new batch of HBS solutions made. Only use HBS solutions with the highest transfection efficiency (at least 80+% of EmGFP- or mRFP-positive cells) for virus production (see section 6.1).