Paul.xu at 23:05, 21 March 2016

2016-03-21T23:05:56Z

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= 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''' ===

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

[[File:2016 3 20 compare.JPG|RTENOTITLE]]

3/21/2016 transfection result

[[File:0321 Transfection.JPG|RTENOTITLE]]

[[File:03212016 transfection.JPG|RTENOTITLE]]

== '''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.
[[Category:Paul]] [[Category:Transfection]]

Synthesis of PHP-B1 - Revision history

Paul.xu at 23:05, 21 March 2016