Paul.xu at 12:37, 24 June 2014

2014-06-24T12:37:04Z

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== Synthesis of PHP ==

1. 0.75 g DAP + 1.8 g DBP + 1.5 ml MeOH, react at 50 oC o/n

2. Wash with 2X 10 ml MeOH, 5 EtOH +5 ml MeOH (should wash 3 X 10 MeOH)

3. Add DMSO to 12 ml--> suspension

4. Make 1g PEI18 + DMSO to final 10 g

== Synthesis of DTBP-PHP ==

1. Make 100 mg DSP and DTBP in  1ml DMSO. DTBP does not completely dissove in DMSO so it is suspension. DSP can dissolve in DMSO

2. Reaction

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Sample #
| C
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
|-
| PHP sol (ml)
| 0.5 
| 0.5 
| 0.5 
| 0.5 
| 0.5
| 0.5
| 0.5
| 0.5
| 0.5
|-
| 0.1g/ml PEI18 (ul)
| 50
| 50
| 50
| 50
| 50
| 50
| 50
| 50
| 50
|-
| 0.1g/ml DSP (ul)
| 
| 50 
| 100 
|  150
| 200
|
|
|
|
|-
| DSP (mg)
| 
|  5
| 10 
| 15 
| 20
|
|
|
|
|-
| 0.1g/ml DTBP (ul)
|
|
|
|
| 
| 50
| 100
| 150
| 200
|-
| DMSO
| 200
| 150
| 100
| 50
|
| 150
| 100
| 50
|
|-
| Observations
| PHP suspension became clear in < 1 min
| PHP suspension became clear in < 1 min
| '''form gel like polymer'''
| '''form gel like polymer'''
| '''form gel like polymer'''
| PHP suspension became clear in < 1 min
| '''form gel like polymer'''
| '''form gel like polymer'''
| '''form gel like polymer'''
|}

Let reaction o/n at room temp.

(6/21/14: The gel like polymer does not have capability to transfect DNA: Spin down polymer, add 10 mM Hepes buffer, pH 7.0. Dilute to 1:10, use 3 ul to transfect 1 ug DNA, no GFP expression)

3. Votex, dilute reaction product in 10 mM HEPES, pH7.0

1:10: 1ul to 19 ul

1:30: 1ul to 29 ul 

1:50 1 ul to 49 ul

4. Do DNA-dye binding exclusion assay

[[Image:TR DNA Dye assay.JPG]]

5. Transfection: 293T/E cells in 24 well plate. 3 ul diluted TR + 25 ul DMEM, add 0.5 ug DNA in 25 ul DMEM, incubate at RT for 20 min. Add to one well (500 ul complete DMEM medium with 10% FBS). Take pictures after o/n transfection.

 

[[Image:DSP vs DTBP conjugation.jpg|400px|DSP vs DTBP conjugation.jpg]]

2nd Day 

[[Image:DTBP DSP PHP 2nd day.jpg]]

 

[[Image:PHP-DSP vs DTBP 6 21 2014.jpg]]

 

[[Image:PHP DSP vs DTBP 2012 6 21-2.JPG]]

6/22/14

[[Image:PHP-DTBP-PEI18 6 22 2014.jpg]]

[[Image:PHP-DTBP-PEI18 6 22 14 2.JPG]]

6/23/14

[[Image:PHP-DSP_vs_DTBP_6_23_2014.JPG]]

[[Image:PHP-DSP_vs_DTBP_6_23_2014_2.JPG]]

6/24/14

[[Image:PHP-DSP_vs_DTBP_6_24_2014.JPG]]

[[Image:PHP-DSP_vs_DTBP_6_24_2014_2.JPG]]

 

Conclusion: DSP is better than DTBP

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

Synthesis PHP-DTBP-PEI18 - Revision history

Paul.xu at 12:37, 24 June 2014