Synthesis of GeneExpresso

Synthesis of GeneExpresso

Synthesis

1. weigh 750 mg SOLID into a glass vial

2, weigh 1.8 g OIL (~910 ul)

3, add 1.5 ml MeOH

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 10 ml, this is called GexnExpresso Stock, put into a glass vial with TEFLON seal cap.

16. Add 0.75 ml of PEI18 stock: 1 g PEI18, 0.5 g pyrozole in 10 ml H2O (100 mg/ml)

The final amount of PEI is 75 mg PEI18

17. Add 75 mg DSP. Mix, put the glass vial into 2 plastic zip-lock bags, close, react with a rotary mixer o/n

18. Add 100 mg pyrazol, react with a rotary mixer at RT for 3~5 hour.

19. Transfer 5 ml to one 50 ml centrifuge tubes, add 40 ml of isopropanol to precipiate polymer. Spin 3000 rpm for 10 min, decant isopropanol.

21. For long term storage: Store the solid pellet (do not add water or buffer) at -20 oC.

22. Add autoclaved ultrapure water 0.22u filter sterilized 250 mM HEPES, pH 7.5 to 10 ml, aliquot 1 ml to autoclaved 1.5 ml microcentrifuge tubes and store them at -20 oC.

23, To make 1 ml final product, aliquot 50 ul to 1.5 ml sterilized tubes. Store at -20 oC freezer. Just before use, add 950 ml ultrapure water before use

24. To make 100 ul free sample, aliquot  5 ul to 0.5 ml sterilized tubes. Store at -20 oC freezer. Just before use, add 950 ml ultrapure water before use

QC

Dilution

Dilution Buffer: Filtered 10 mM HEPES, PH 7.2 or water

	GexnExpresso Stock (ul)	Dilution Buffer (ul)	Dilution
1	15	985	1:66.7
2	20	980	1:50
3	40	960	1:25
4	50	950	1:20
5	75	925	1:13.3

Dilute using a 96 well plate: Final vol is 200 ul

#	GexnExpresso Stock (ul)	Dilution Buffer (ul)	Dilution
1	3	197	1:66.7
2	4	196	1:50
3	8	192	1:25
4	10	190	1:20
5	15	185	1:13.3

Transfection 293T cells in 24 well plate, transfect with 1 ug  DNA (GFP) + 3 ul diluted transfection reagent in 25 ul of serum-free DMEM. Positive control is Lipofectamine 2000. Check transfection efficiency and cell viability next day, take a picture under fluorescence microscopy

Product

Find the best dilution, (usually 1:20 dilution is the best, i.e., 50 ul stock to 950 ul water),  use this dilution for making transfection reagent, filter through a 0.22 u filter to sterize, aliquot (1 ml) and store at -20 oC.