Paul.xu at 18:01, 29 July 2014

2014-07-29T18:01:40Z

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== 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. --> this is CS stock.

7/22/14 

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

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

reaction RT o/n

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

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

 

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

== Conjugate Chitosan to PHP/PEI ==

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

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

pticture of 7/25/14

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

7/26/14 

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

7/27/14 

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

7/29/14

[[Image:Chitosan_PHP_AG_7_29_14.JPG]]

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

Synthesis Chitosan Polyamine - Revision history

Paul.xu at 18:01, 29 July 2014