Editing Synthesis Chitosan Polyamine

== Conjugate CS with PEI  ==

0.25 g Chitosan from polyscience, + 25 ml H2O, + 0.1 ml H2SO4, autoclave 30 min, cool down, add 0.15M NaOH to 50 ml, spin, wash ppt 2 X with H2O, spin, dissolve pellet in 20 ml 0.2 N HoAc, adjust pH to 6, spin to remove insoluble stuff, filter. --&gt; this is CS stock. 

7/22/14<br> 

{| width="563" cellspacing="1" cellpadding="1" border="1"
|-
| <br> 
| CS<br> 
| Polyamine<br> 
| 8N HoAc<br> 
| Linker<br> 
| Observation<br>
|-
| 1<br> 
| 20 ul<br> 
| 2 ul 0.4 mg/ml PEI18<br> 
| 1ul<br> 
| <br> 
| <br>
|-
| 5<br> 
| 20 ul&nbsp;&nbsp; <br> 
| 2 ul 0.4 mg/ml PEI18<br> 
| 1ul<br> 
| 5 ul 100mg/ml DTBP<br> 
| <br>
|-
| 2<br> 
| 20 ul 
| 2 ul 0.5 ug/ul agmatine<br> 
| <br> 
| <br> 
| insoluble ppt<br>
|-
| 6<br> 
| 20 ul 
| 2 ul 0.5 ug/ul agmatine<br> 
| <br> 
| 5 ul 100mg/ml DTBP<br> 
| insoluble ppt<br>
|-
| 3<br> 
| 20 ul 
| 2ul PHP stock in DMSO<br> 
| <br> 
| <br> 
| <br>
|-
| 7<br> 
| 20 ul 
| 2ul PHP stock in DMSO<br> 
| <br> 
| 5 ul 100mg/ml DTBP<br> 
| <br>
|-
| 4<br> 
| 20 ul 
| 2ul PHP conc in DMSO, 2ul 0.4 mg/ml PEI18<br> 
| 1ul<br> 
| <br> 
| <br>
|-
| 8<br> 
| 20 ul 
| 2ul PHP conc in DMSO, 2 ul 0.4 mg/ml PEI18<br> 
| 1ul<br> 
| 5 ul 100mg/ml DTBP<br> 
| <br>
|-
| 9<br> 
| 20 ul&nbsp; 
| 2 ul 0.5 ug/ul agmatine<br> 
| <br> 
| 5 ul 100mg/ml DSP<br> 
| insoluble ppt<br>
|-
| 0 (control)<br> 
| 20 ul <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

PEI18 will ppt CS, but add 1 ul 8N HoAc will dissolve CS (adjust pH back to ~6).<br> 

reaction RT o/n 

7/23/14 dilute 1:20 with water, do DNA dye exlusion assay 

[[Image:Chitosan conjugate 7 23 14.JPG]] 

<br> 

Dilute 1:20 with 10 mM Hepes, pH7.0, transfection: 1 ug DNA. 24 well plate 293T cells 

== Conjugate Chitosan to PHP/PEI<br>  ==

{| width="756" cellspacing="1" cellpadding="1" border="1"
|-
| Plate Pos<br> 
| Sample #<br> 
| CS sol<br> 
| Conjugate<br> 
| Linker<br> 
| Observation<br>
|-
| 2<br> 
| 1<br> 
| 20 ul<br> 
| 2 ul 0.4 mg/ml PEI18<br> 
| <br> 
| rowspan="2" | need to add 1 ul 8N HOAc to adjust to pH&lt;7 to dissolve CS<br>
|-
| 3<br> 
| 5<br> 
| 20&nbsp;&nbsp;&nbsp;&nbsp; <br> 
| 2 ul 0.4 mg/ml PEI18<br> 
| 5 ul 100 mg/ml DTBP<br>
|-
| 4<br> 
| 2<br> 
| 20 
| 2 ul 0.5 mg/ml agmatine<br> 
| <br> 
| rowspan="2" | &nbsp;form insoluble ppt<br>
|-
| 5<br> 
| 6<br> 
| 20 
| 2 ul&nbsp; 0.5 mg/ml agmatine<br> 
| 5 ul 100 mg/ml DTBP<br>
|-
| 6<br> 
| 3<br> 
| 20 
| 2 ul PHP in DMSO<br> 
| <br> 
| <br>
|-
| 7<br> 
| 7<br> 
| 20 
| 2 ul PHP in DMSO<br> 
| 5 ul 100 mg/ml DTBP<br> 
| <br>
|-
| 8<br> 
| 4<br> 
| 20 
| 2 ul PHP in DMSO + 2 ul 0.4 mg/ml PEI18 <br> 
| <br> 
| rowspan="2" | <br>need to add 1 ul 8N HOAc to adjust to pH&lt;7 to dissolve CS
|-
| 9<br> 
| 8<br> 
| 20&nbsp; <br> 
| 2 ul PHP in DMSO + 2 ul 0.4 mg/ml PEI18 
| 5 ul 100 mg/ml DTBP<br>
|-
| 10<br> 
| 9<br> 
| 20 <br> 
| 2 ul 0.5 mg/ml agmatine<br> 
| 2 ul 0.5 mg/ml agmatine<br> 
| <br>
|-
| 1<br> 
| C<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 11<br> 
| PHP + AG +&nbsp;DSP<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 12<br> 
| PHP + AG + DTBP<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 13<br> 
| PHP + AG, ppt<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 14<br> 
| PHP + AG, oil<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 15<br> 
| 7/19/14, PHP+DSP-PEI18, purified 1:10<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 16<br> 
| 7/19/14, PHP+DSP-PEI18, purified 1:30<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 17<br> 
| 7/19/14, PHP+DSP-PEI18, unpurified 1:10<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 18<br> 
| 7/19/14, PHP+DSP-PEI18, unpurified 1:30<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 19<br> 
| 7/20/14, PHP+DSP-PEI18, 1:10<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 20<br> 
| 7/20/14, PHP+DSP-PEI18, 1:30<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 21<br> 
| L2K<br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

7/23/14: Transfection: dilute 1 ul to 20 ul. use 3 ul to transfect 293T cells<br> 

pticture of 7/25/14 

[[Image:Chitosan PHP AG 7 25 14.JPG]]<br> 

7/26/14<br> 

[[Image:Chitosan PHP AG 7 26 14.JPG]]<br> 

7/27/14<br> 

[[Image:Chitosan PHP AG 7 27 14.JPG]] 

7/29/14

[[Image:Chitosan_PHP_AG_7_29_14.JPG]]

[[Category:Transfection]] [[Category:Paul]]