Synthesis Chitosan Polyamine
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Conjugate CS with PEI
[edit]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
| 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
Dilute 1:20 with 10 mM Hepes, pH7.0, transfection: 1 ug DNA. 24 well plate 293T cells
Conjugate Chitosan to PHP/PEI
[edit]| Plate Pos |
Sample # |
CS sol |
Conjugate |
Linker |
Observation |
| 2 |
1 |
20 ul |
2 ul 0.4 mg/ml PEI18 |
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 |
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 |
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
7/26/14
7/27/14
7/29/14
