OhioMod2013:Methods/Liposomes: Difference between revisions
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==Encapsulation Efficiency== | ==Encapsulation Efficiency== | ||
*Add 4 μL of 1mg/ml lipid (or 2μL of 2mg/ml conc.) to 10 μL of 245 ng of DNA origami in | *Add 4 μL of 1mg/ml lipid (or 2μL of 2mg/ml conc.) to 10 μL of 245 ng of DNA origami in 10 mM citric buffer with 40% ethanol. Make two of these. | ||
*Also make two aliquots of 4 μL (or 2 ul) of lipid in 100 ul of FOBM16 with 5 nM staples present. | *Also make two aliquots of 4 μL (or 2 ul) of lipid in 100 ul of FOBM16 with 5 nM staples present. | ||
*Allow to equilibrate for 15 minutes. | *Allow to equilibrate for 15 minutes. | ||
*Add Yoyo1 at 10:1 base-pair/yoyo ratio. Since we're assuming 5 nM origami of 1450 base-pairs in 10 μL, then add 0.1450 nM of YOYO into solution. | *Add Yoyo1 at 10:1 base-pair/yoyo ratio. Since we're assuming 5 nM origami of 1450 base-pairs in 10 μL, then add 0.1450 nM of YOYO into solution. | ||
* | *Take Flourescence with 488 nm channel for step 1. | ||
*Take Flourescence with 488 | *Add 2 ul of 0.1% igepal detergent to break up the liposomes. Make sure to pipet up and down to mix up the detergent. | ||
*Take Flourescence with 488 nm channel for step 2. | |||
[[Image:Yoyo-1.jpg]] | [[Image:Yoyo-1.jpg]] | ||
Latest revision as of 16:06, 4 July 2013
Got liposomes from Bryant Yung of Dr. Lee's lab. Two tubes of:
- QTCN: Quaternary-Tertiary Lipid Amine Cationic Nanoparticles (1 mg/mL)
- SPLN: Small Peptide Lipid Nanoparticles (2 mg/mL)
Kept in 40% ethanol in 10 mM citric acid buffer (pH of 5). The lipid mixture is composed of DODAP, Lac-DOPE, DOPE, DMG-PEG, and with or without gramacidin A, at a molar ratio of 50:10:28:2:10 respectively.
Combine with DNA origami
- Take lipid stock, combine with oligo at ratio of 1:15 DNA/lipid(weight/weight) in 10x transfection medium.
- Set aside 10-15 min to allow for electrostatic interaction.
- Dilute to final volume
- Extrude through polycarbonate filter, or other sterile filter in general
Encapsulation Efficiency
- Add 4 μL of 1mg/ml lipid (or 2μL of 2mg/ml conc.) to 10 μL of 245 ng of DNA origami in 10 mM citric buffer with 40% ethanol. Make two of these.
- Also make two aliquots of 4 μL (or 2 ul) of lipid in 100 ul of FOBM16 with 5 nM staples present.
- Allow to equilibrate for 15 minutes.
- Add Yoyo1 at 10:1 base-pair/yoyo ratio. Since we're assuming 5 nM origami of 1450 base-pairs in 10 μL, then add 0.1450 nM of YOYO into solution.
- Take Flourescence with 488 nm channel for step 1.
- Add 2 ul of 0.1% igepal detergent to break up the liposomes. Make sure to pipet up and down to mix up the detergent.
- Take Flourescence with 488 nm channel for step 2.
