preparation of lipoplex

Preparation of 10 ml of 20 mM DODAP:Chol (1:1)

1. preparation

DOTAP (Avanti, 890890)

cholesterol (Avanti,700000)

rotary water bath

D5W (5% Dextrose in water)

sonicator

filter

2. protocol

3. Warm-up DOTAP at RT and weigh DOTAP 140 mg (0.2 mmol) (MW = 693.55, Cat#: 890890, 1g and 1:18)

4. Wight Chol. 77 mg (0.2 mmol) (MW = 386.66, Cat# 700000, >98% pure)
5. Add them to a clean round-bottom flask (1 L) respectively and then cover the flask with parafilm.
6. Add 10 mL HPLC-graded CHCl3 to the bottle and be sure the chemicals are all dissolved.
7. Load the flask on the rotary water bath at 30 C for 30 min under vacuum at about 30 Hg (the pump Model B-169 is made by the Brinkman). At this stage the thin layer of film is formed.
8. Lyophilize the dried film to get rid of the trace amount of chloroform for 15 min.
9. Hydrate the thin film in the round flask with 10 ml of D5W (5% Dextrose in water) in hood to give a final concentration of 20 mM DOTAP:Chol.
10. Pre-wet everywhere of the film in the hood as quickly as possible and use parafilm to cover the flask and punch several small holes on the cover with needle.
11. Hold the flask in water bath at 50 ْC and rotate at different angles for at least 15 min or until you see most of the material is off the flask and then vertically move the flask for another 30 min.
12. Transfer the flask into a 37 ْC water bath and vertically move the flask for 10 min.
13. Cover the flask and leave it over-night at RT.
14. Hold the flask with a clamp on a stand and place it into a 50 ْC water bath sonicator and sonicate the DOTAP mixture at low frequency (low f = 35 kHz, high = 80 kHz) at 50 ْC for 5 min.
15. Bring the flask into a hood and carefully transfer the product into a 15 mL tube using 10 mL pipette.
16. Warm-up the mixture in the tube in a water bath at 50 ْC for 10-12 min.
17. Meanwhile open all 10 mL syringes in the hood, each syringe with 1.0, 0.45, 0.2, and 0.1 mm filter disk (Whatman Filters, Kent, UK), respectively, on it.
18. Take the tube out of water bath and, sequentially, filtrate the mixture into another syringe as quickly as possible.
19. Collect the DOTAP solution from the last filtration into the glass tube, about 1 drop/色c.
20. Transfer the final product into a 1.5 mL glass vials to fill all the space and cap the vial and store at 4 ْC.
21. O.D. the products: dilute 5 ml of DOTAP:Chol into 95 ml f dH2O, measure O.D. at 400 nm (vis). The normal range of O.D. should be in 0.35-0.45.