Gu PHP-DSP-PEI18
Gu of PHP-DSP-PEI18
[edit]| # | PHP | 0.4mg/ul PEI18 | 10mg/ml DSP | 0.5ug/ul GU | DMSO | Purify by Ethanol ppt |
| 1 | 50 ul | 5 ul | 7.5 ul |
0 |
12.5 |
n |
| 2 | 50 ul | 5 |
7.5 |
2.5 |
10 |
n |
| 3 | 50 ul | 5 |
7.5 | 5 |
7.5 |
n |
| 4 | 50 ul | 5 |
7.5 | 7.5 |
5 |
n |
| 5 | 50 ul | 5 |
7.5 | 10 |
2.5 |
n |
| 6 | 50 ul | 5 |
7.5 | 12.5 |
0 |
n |
| 7 | 50 ul | 5 |
7.5 | 0 |
12.5 |
y |
| 8 | 50 ul | 5 |
7.5 | 2.5 |
10 |
y |
| 9 | 50 ul | 5 |
7.5 | 5 |
7.5 |
y |
| 10 | 50 ul | 5 |
7.5 | 7.5 |
5 |
y |
| 11 | 50 ul | 5 |
7.5 | 10 |
2.5 |
y |
| 12 | 50 ul | 5 |
7.5 | 12.5 |
0 |
y |
Add 50 ul PHP in DMSO, 5 ul 0.4 mg/ul PEI18 in DMSO, mix, add 7.5 ul DSP in DMSO, incubate at RT o/n
6/30/14, add pyrozole (0.5ug/ul) incubate at RT ~ 3 hr
purification with ethanol: add 10 volume of ethanol, spin remove sup. Add DMSO to original volume to dissolve white ppt.
take 1 ul and diluted with Hepes buffer (10 mM), pH 7.0, use 3 ul diluted TR + 1 ug DNA to transfect 293T cells on 24 wells plate
take a picture 2 days after transfection (7/2/14)
6/30/14: Transfect use 1:30 dilution
[edit]Picture of 7/2/14
Picture of 7/3/14
Picture of 7/4/14
picture of 7/5/14
picture of 7/6/14
7/3/14 Transfect use 1:10 dilution
[edit]Picture of 7/4/14
picture of 7/5/14
picture: 7/6/14
picture 7/8/14
Gu of PHP-DSP-PEI18 vs PHP-DSP-GuPEI18
[edit]compare pre-Gu or Post-Gu
| # | PHP | 0.4mg/ul PEI18 |
Gu-PEI18 (5 ul of 0.4 mg/ul PEI18 +0.5ug/ul GU ) |
10mg/ml DSP | 0.5ug/ul GU |
DMSO | Purify by Ethanol ppt |
| 1 | 50 ul | 5 ul | 7.5 ul |
0 |
12.5 |
n | |
| 2 | 50 ul | 5 |
7.5 |
2.5 |
10 |
n | |
| 3 | 50 ul | 5 |
7.5 | 5 |
7.5 |
n | |
| 4 | 50 ul | 5 |
7.5 | 7.5 |
5 |
n | |
| 5 | 50 ul | 5 |
7.5 | 10 |
2.5 |
n | |
| 6 | 50 ul | 5 |
7.5 | 12.5 |
0 |
n | |
| 7 | 50 ul | 5 ul + 0ul |
7.5 | 12.5 |
n | ||
| 8 | 50 ul | 5 ul + 2.5 ul |
7.5 | 10 |
n | ||
| 9 | 50 ul | 5 + 5 |
7.5 | 7.5 |
n | ||
| 10 | 50 ul | 5 + 7.5 |
7.5 | 5 |
n | ||
| 11 | 50 ul | 5 + 10 |
7.5 | 2.5 |
n | ||
| 12 | 50 ul | 5+ 12.5 |
7.5 | 0 |
n |
7/3/2014: use 1: 30 and 1:10 diluted TR for transfection. 293 cells on 24 wells plate, 1 ug DNA per well
picture of 7/4/2014
picture of 7/5/14
picture 7/6/14
Picture of 7/8/14
7/9/14
7/10/14
7/11/14
7/12/14
7/13/14
Stability
[edit]Dilute polymer PHP-Gu-PEI18 with DMSO or 10 mM HEPES, pH7.0 to make 1:10 stock.
Heat at 50 oC for 2 days.
Use 1:10 stock or 3-fold dilution (diluted 3 folds) for transfection
| Sample | Transfection Dilution | Heat treatment | Storage solvent | |
| No heat | ||||
| 1 | 6/24/14#1 |
1:30 |
n | H2O |
| 7 | 6/24/14#1 |
1:30 | n | DMSO |
| 13 | 6/24/14#1 | 1:10 | n | H2O |
| 19 | 6/24/14#1 |
1:10 |
n | DMSO |
| 2 | 6/24/14#5P | 1:30 | n | H2O |
| 8 | 6/24/14#5P | 1:30 | n | DMSO |
| 14 | 6/24/14#5P |
1:10 | n | H2O |
| 20 | 6/24/14#5P |
1:10 | n |
DMSO |
| 3 | 7/3/14#1 |
1:30 | n |
H2O |
| 9 | 7/3/14#1 |
1:30 | n |
DMSO |
| 15 | 7/3/14#1 |
1:10 | n |
H2O |
| 21 | 7/3/14#1 |
1:10 | n |
DMSO |
| 4 | 7/3/14#2 |
1:30 | n |
H2O |
| 10 | 7/3/14#2 | 1:30 | n |
DMSO |
| 16 | 7/3/14#2 | 1:10 | n |
H2O |
| 22 | 7/3/14#2 | 1:10 | n |
DMSO |
| heat at 50 oC for two days | ||||
| 5 | 6/24/14#1 |
1:30 |
y |
H2O |
| 11 | 6/24/14#1 | 1:30 | y |
DMSO |
| 17 | 6/24/14#1 | 1:10 | y |
H2O |
| 23 | 6/24/14#1 | 1:10 | y |
DMSO |
| 6 | 6/24/14#5P |
1:30 | y |
H2O |
| 12 | 6/24/14#5P | 1:30 | y |
DMSO |
| 18 | 6/24/14#5P | 1:10 | y |
H2O |
| 24 | 6/24/14#5P | 1:10 | y |
DMSO |
| 25 | 7/3/14#1 |
1:30 | y |
H2O |
| 27 | 7/3/14#1 | 1:30 | y |
DMSO |
| 29 | 7/3/14#1 | 1:10 | y |
H2O |
| 31 | 7/3/14#1 | 1:10 | y |
DMSO |
| 26 | 7/3/14#2 |
1:30 | y |
H2O |
| 28 | 7/3/14#2 | 1:30 | y |
DMSO |
| 30 | 7/3/14#2 | 1:10 | y |
H2O |
| 32 | 7/3/14#2 | 1:10 | y |
DMSO |
| L3K | ||||
| Turbofect | ||||
7/9/14
7/10/14
7/11/14
7/12/14
7/13/14
File:PHP GU PEI stability 7 13 14 3.JPG
7/14/14
Stablity Test 2
[edit]7/10/14
Dilute polymer PHP-Gu-PEI18 with DMSO or 10 mM HEPES, pH7.0 to make 1:10 stock.
Heat at 50 oC for 4 days.
Use 1:10 stock for transfection, 293T 24 wells, 1ug DNA, 3 ul TR
| Sample # |
6/24/14 sample #1 |
6/24/14 sample #5P (purified by ethano ppt) |
7/3/14#1 |
7/3/14#2 |
Storage Buffer: 10 mM HEPES, pH7 |
Storage: DMSO |
| 1 |
* |
* |
||||
| 2 |
* |
* | ||||
| 3 |
* |
* |
||||
| 4 |
* |
* | ||||
| 5 |
* |
* |
||||
| 6 |
* |
* | ||||
| 7 |
* |
* |
||||
| 8 |
* |
* |
Picture of 7/12/14
7/13/14
7/14/14
7/16/14
7/17/14
7/18/14
7/19/14
7/20/14
Stability Test 3
[edit]incubate polymers at 50 oC for 7 days, other conditions are as Stability Test 2
transfection is on 7/14/14
picture of 7/16/14 7/14/14
7/17/14
7/18/14
7/19/14
7/20/14
7/10/14
Dilute polymer PHP-Gu-PEI18 with DMSO or 10 mM HEPES, pH7.0 to make 1:10 stock.
Heat at 50 oC for 4 days.
Use 1:10 stock for transfection, 293T 24 wells, 1ug DNA, 3 ul TR
Stability Test 5
[edit]8/1//14
Stability Test Samples, has been stored at 50 oC for > 2 weeks
| Sample # |
6/24/14 sample #1 |
6/24/14 sample #5P (purified by ethano ppt) |
7/3/14#1 |
7/3/14#2 |
Storage Buffer: 10 mM HEPES, pH7 |
Storage: DMSO |
| 1 |
* |
* |
||||
| 2 |
* |
* | ||||
| 3 |
* |
* |
||||
| 4 |
* |
* | ||||
| 5 |
* |
* |
||||
| 6 |
* |
* | ||||
| 7 |
* |
* |
||||
| 8 |
* |
* |
8/1//14 Transfection
1 ug DNA, 293T cells in 24 well plate
| Well |
Sample |
Description |
Note |
| 1 |
PHP-DSP-AG |
||
| 2 |
PHP-DTBP-AG |
||
| 3 |
PHP-DSP-AG, ppt |
||
| 4 |
PHP-DSP-AG, oil |
||
| 5 |
K-DBP-AG |
Lysine + DBP + AG |
very weak DNA binding |
| 6 |
K-CBA-AG |
Lysine + CBA + AG |
very weak DNA binding |
| 7 |
PHP-DSP-AG, 6/15/14 |
6/15/14 PHP | |
| 8 |
PHP-DTBP-AG, 6/15/14 |
6/15/14 PHP | |
| 9 |
PHP-DSP-AG, 7/17/14 |
7/17/14 PHP | |
| 10 |
PHP-DTBP-AG, 7/17/14 |
7/17/14 PHP | |
| 11 |
Stability #1 |
Store at RT |
|
| 12 |
Stability #1 |
Heat 50 oC |
|
| 13 |
Stability #2 |
Store at RT | |
| 14 |
Stability #2 | 50 oC |
|
| 15 |
Stability #3 | Store at RT | |
| 16 |
Stability #3 | 50 oC | |
| 17 |
Stability #4 | Store at RT | |
| 18 |
Stability #4 | 50 oC | |
| 19 |
Stability #5 | Store at RT | |
| 20 |
Stability #5 | 50 oC | |
| 21 |
Stability #6 |
Store at RT | |
| 22 |
Stability #6 | 50 oC | |
| 23 |
Stability #7 | Store at RT | |
| 24 |
Stability #7 | 50 oC |
Picture of 8/2/14
8/3/14
8/4/14
8/5/14
8/3/14
| Well |
Sample |
||
| 1 |
Stability #2 |
Store at RT |
|
| 2 |
Stability #2 |
50 oC |
|
| 3 |
Stability #8 |
Store at RT |
|
| 4 |
Stability #8 |
50 oC |
|
| 5 |
7/17/14 PHP conjugated to PEI18 with DSP |
1:10 |
|
| 6 |
7/17/14 PHP conjugated to PEI18 with DSP |
1:30 |
|
| 7 |
7/17/14 PHP conjugated to PEI18 with DSP |
1:10 |
|
| 8 |
7/17/14 PHP conjugated to PEI18 with DSP |
1:30 |
|
| 9 |
Stability #1 |
Store at RT |
Picture of 8/5/14
