Editing Synthesis of GeneExpresso

= <span dir="auto">Synthesis of GeneExpresso</span> =

== '''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>&nbsp;0.22u filter&nbsp;sterilized&nbsp;250&nbsp;mM HEPES, pH 7.5&nbsp;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&nbsp;tubes. Store at -20 oC freezer. Just before use, add 950 ml ultrapure water before use

24. To make 100 ul free sample, aliquot &nbsp;5 ul to 0.5 ml sterilized&nbsp;tubes.&nbsp;<span style="line-height: 20.8px;">Store at -20 oC freezer. Just before use, add 950 ml ultrapure water before use</span>

== '''QC''' ==

=== '''Dilution''' ===

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

{| style="width: 500px" border="1" cellpadding="1" cellspacing="1"
|-
| <br/>
| GexnExpresso Stock (ul)<br/>
| Dilution Buffer (ul)<br/>
| Dilution<br/>
|-
| 1<br/>
| 15<br/>
| 985<br/>
| 1:66.7<br/>
|-
| 2<br/>
| 20<br/>
| 980<br/>
| 1:50<br/>
|-
| 3<br/>
| 40<br/>
| 960<br/>
| 1:25<br/>
|-
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">4</span></span><br/>
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">50</span></span><br/>
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">950</span></span><br/>
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">1:20</span></span><br/>
|-
| 5<br/>
| 75<br/>
| 925<br/>
| 1:13.3<br/>
|}

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

{| border="1" cellpadding="1" cellspacing="1"
|-
| #<br/>
| GexnExpresso Stock (ul)<br/>
| Dilution Buffer (ul)<br/>
| Dilution<br/>
|-
| 1<br/>
| 3<br/>
| 197<br/>
| 1:66.7<br/>
|-
| 2<br/>
| 4<br/>
| 196<br/>
| 1:50<br/>
|-
| 3<br/>
| 8<br/>
| 192<br/>
| 1:25<br/>
|-
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">4</span></span><br/>
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">10</span></span><br/>
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">190</span></span><br/>
| <span style="color:#FFFF00"><span style="background-color: rgb(255, 0, 0)">1:20</span></span><br/>
|-
| 5<br/>
| 15<br/>
| 185<br/>
| 1:13.3<br/>
|}

===  ===

===  ===

'''Transfection''' 293T cells in 24 well plate, transfect with 1 ug&nbsp; 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&nbsp;ul water),&nbsp; 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]]