Synthesis of PHP-B1

Synthesis of GeneExpresso

Protocol 1

1. weigh 750 mg SOLID into a glass vial.

2, add 1 ml MeOH, dissolve

3, weigh 1.8 g OIL (add ~910 ul)

4, mix

5, add 300 ul (300mg) TREN to 1 ml MeOH, add to the above mixture

6, mix, tighten the cap

7, incubate inside an rotary oven for 2 days, temperature is 50 oC

8, add ~5 ml ddH2O to dissolve

9, add 45 ml isopropanol, mix

10, spin for 5 min at 3000 rpm

11, discard the liquid

12, add ~ 5ml ddH2O to dissolve

13, add 45 ml isopropanol, mix

14, spin for 5 min at 3000 rpm

15, discard the liquid

16, add ddH2O to 10 ml, this is called GexnExpresso (PHP) Stock

17, store at -20 oC.

Protocol 2

1. weigh 750 mg SOLID into a glass vial

2, add 2 ml MeOH, dissolve

3, weigh 1.8 g OIL (~900 ul)

4, dissolve

5, mix, tighten the cap

6, incubate inside an rotary oven for 2 days, temperature is 50 oC

7, add ~5 ml ddH2O to dissolve

8, add 45 ml isopropanol, mix

9, spin for 5 min at 3000 rpm

10, discard the liquid

11, add ~ 5ml ddH2O to dissolve

12, add 45 ml isopropanol, mix

13, spin for 5 min at 3000 rpm

14, discard the liquid

15, add ddH2O to 15 ml, this is called GexnExpresso (PHP-B1) Stock

16, store at -20 oC.

QC

Dilution

	GexnExpresso Stock (ul)	Dilution Buffer (ul)	Dilution
1	10	990	1:100
2	15	985	1:66.7
3	20	980	1:50
4	30	970	1:33
5	40	960	1:25
6	50	950	1:20

Transfection

293T cells in 24 well plate, transfect with 1 ug  DNA (GFP) + 3ul diluted transfection reagent. Check transfection efficiency and cell viability next day, take a picture under fluorescence microscopy

3/20/2016 transfection result

3/21/2016 transfection result

Product

Find the best dilution, use this dilution for making transfection reagent, for example, add 2 ml stock to 50 ml, filter through a 0.22 u filter to sterize, aliquot (1 ml) and store at -20 oC.