Synthesis PHP-DTBP-PEI18

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

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

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.

2nd Day

6/22/14

6/23/14

6/24/14

Conclusion: DSP is better than DTBP