Biolistic-Mediated Gene Transfer: A Technique to Deliver Gene of Interest in Target Cells via a Biolistic Gene Gun

Samenvatting

Source: Taylor, T., et al. Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans. J. Vis. Exp. (2015).

In this video, we demonstrate the protocol of biolistic gene gun-mediated gene delivery in cultured cells. This method provides a simple method of stable genetic transformation using a minimal quantity of DNA.

Protocol

1. DNA Preparation

1. Prepare orange macrocarrier biolistic discs by submerging them in 100% ethanol using forceps. Place discs into a large petri dish containing drierite to dry (make sure the drierite does not touch the discs).
2. Once dry, press the macrocarrier discs into the silver disc holders (previously wiped down with 100% ethanol).
3. Vortex gold microcarrier beads suspended in 100% ethanol and aliquot 12 µl into a 1.5 ml microcentrifuge tube, one tube per transformation.
4. Add to each tube in order: 2 µg of DNA (preferably 2 µl of 1 µg/µl of DNA), 10 μl 2.5 M CaCl₂, and....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
0.6 μm gold beads	Bio-Rad	165-2262	http://www.bio-rad.com
Spermadine-free base	Sigma- Aldrich	S0266	https://www.sigmaaldrich.com
1350 psi Rupture Discs	Bio-Rad	165-2330	http://www.bio-rad.com
Stopping Screens	Bio-Rad	165-2336	http://www.bio-rad.com
Macrocarriers discs	Bio-Rad	165-2335	http://www.bio-rad.com
PDS-1000/He System	Bio-Rad	165-2257	http://www.bio-rad.com
Microfuge 18 Centrifuge	Beckman Coulter	F241.5P	www.beckmancoulter.com