Paul.xu at 19:39, 5 August 2014

2014-08-05T19:39:15Z

New page

== Gu of PHP-DSP-PEI18 ==

{| cellspacing="1" cellpadding="1" border="1" width="709"
|-
| #
| 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 ===

Picture of 7/2/14 

[[Image:PHP PEI18 GU 2014 7 2.JPG]]

Picture of 7/3/14 

[[Image:PHP PEI18 GU 2014 7 3.JPG]]

Picture of 7/4/14 

[[Image:GU 7 4 14.JPG]] 

picture of 7/5/14 

 

[[Image:PHP-DSP-PEI18 2014 7 5 2.JPG]]

picture of 7/6/14 

[[Image:PHP-DSP-PEI18 2014 7 6 2.JPG]]

 

=== 7/3/14 Transfect use 1:10 dilution ===

Picture of 7/4/14

[[Image:Gu 2014 7 4 2.JPG]]

picture of 7/5/14 

[[Image:PHP-DSP-PEI18 2014 7 5.JPG]] 

picture: 7/6/14

[[Image:PHP-DSP-PEI18 2014 7 6 3.JPG]] 

picture 7/8/14

[[Image:PHP-DSP-PEI18 2014 7 8.JPG]]

== Gu of PHP-DSP-PEI18 vs PHP-DSP-GuPEI18 ==

compare pre-Gu or Post-Gu

{| cellspacing="1" cellpadding="1" border="1" width="709"
|-
| #
| 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

[[Image:Gu 2014 7 4.JPG]]

picture of 7/5/14

[[Image:PHP-DSP-PEI18 2014 7 5 3.JPG]]

picture 7/6/14

[[Image:PHP-DSP-PEI18 2014 7 6.JPG]]

Picture of 7/8/14

[[Image:PHP-DSP-PEI18 2014 7 8 2.JPG]] 

7/9/14 

[[Image:PHP-DSP-PEI18 2014 7 9.JPG]]

7/10/14

[[Image:PHP-DSP-PEI18 2014 7 10.JPG]]

7/11/14 

[[Image:PHP-DSP-PEI18 2014 7 11.JPG]] 

7/12/14 

[[Image:PHP-DSP-PEI18 2014 7 12.JPG]] 

7/13/14

[[Image:PHP-DSP-PEI18 2014 7 13.JPG]]

== Stability ==

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

{| cellspacing="1" cellpadding="1" border="1" width="514"
|-
| 
| Sample
| Transfection Dilution
| Heat treatment
| Storage solvent
|-
| colspan="5" | 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
|-
| colspan="5" | 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
| 
| 
| 
| 
|}

 

[[Image:PHP GU PEI stability 7 8 14.JPG]][[Image:PHP GU PEI stability 7 8 14 2.JPG]]

7/9/14 

[[Image:PHP GU PEI stability 7 9 14 2.JPG]][[Image:PHP GU PEI stability 7 9 14.JPG]]

7/10/14

[[Image:PHP GU PEI stability 7 10 14.JPG]][[Image:PHP GU PEI stability 7 10 14 2.JPG]]

7/11/14 

[[Image:PHP GU PEI stability 7 11 14.JPG]][[Image:PHP GU PEI stability 7 11 14 2.JPG]]

7/12/14 

[[Image:PHP GU PEI stability 7 12 14.JPG]][[Image:PHP GU PEI stability 7 12 14 2.JPG]] 

7/13/14

[[Image:PHP GU PEI stability 7 13 14.JPG]][[Image:PHP GU PEI stability 7 13 14 3.JPG]]

7/14/14 

=== [[Image:PHP GU PEI stability 7 14 14 2.JPG]][[Image:PHP GU PEI stability 7 14 14.JPG]] ===

=== Stablity Test 2 ===

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 

 

{| cellspacing="1" cellpadding="1" border="1" width="628"
|-
| 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

[[Image:PHP GU PEI stability 7 12 14 3.JPG]] 

7/13/14

[[Image:PHP GU PEI stability 7 13 14 2.JPG]]

7/14/14 

[[Image:PHP GU PEI stability 7 14 14 3.JPG]] 

7/16/14 

[[Image:PHP GU PEI stability 7 16 14 2.JPG]] 

7/17/14

[[Image:PHP GU PEI stability 7 17 14 2.JPG]]

7/18/14

[[Image:PHP GU PEI stability 7 18 14 2.JPG]]

7/19/14

[[Image:PHP GU PEI stability 7 19 14.JPG]]

7/20/14

[[Image:PHP GU PEI stability 7 20 14 1.JPG]] 

=== Stability Test 3 ===

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 

[[Image:PHP GU PEI stability 7 16 14.JPG]] 

7/17/14

[[Image:PHP GU PEI stability 7 17 14.JPG]]

7/18/14

[[Image:PHP GU PEI stability 7 18 14.JPG]]

 

7/19/14

[[Image:PHP GU PEI stability 7 19 14 2.JPG]]

7/20/14

[[Image:PHP GU PEI stability 7 20 14.JPG]]

 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 ===

8/1//14 

Stability Test Samples, has been stored at 50 oC for > 2 weeks 

{| cellspacing="1" cellpadding="1" border="1" width="628"
|-
| 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 

{| cellspacing="1" cellpadding="1" border="1" width="542"
|-
| 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 

[[Image:PHP-AG and stability 8 2 14 1.JPG]] 

8/3/14

[[Image:PHP-AG and stability 8 3 14.JPG]]

8/4/14

[[Image:PHP-AG and stability 8 3 14.JPG]]

8/5/14 

 

[[Image:PHP-AG_and_stability_8_5_14.JPG]] 

 

8/3/14 

{| cellspacing="1" cellpadding="1" border="1" width="517" height="277"
|-
| 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 

[[Image:PHP-AG_and_stability_8_5_14_1.JPG]] 

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

Gu PHP-DSP-PEI18 - Revision history

Paul.xu at 19:39, 5 August 2014