Paul.xu at 22:16, 1 July 2016

2016-07-01T22:16:44Z

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= 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 <s>autoclaved ultrapure water</s> 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

{| style="width: 500px" border="1" cellpadding="1" cellspacing="1"
|-
| 
| 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

{| border="1" cellpadding="1" cellspacing="1"
|-
| # 
| 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

=== Untreated, -, + ===

== '''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.
[[Category:Protocols]]

Synthesis of GeneExpresso - Revision history

Paul.xu at 22:16, 1 July 2016