Particle bombardment using biolistic gun
(The protocol is described as such it is performed in Plant Transformation Lab)
Preparation of DNA for PDS 1000/He gene gun:
This gun was originally constructed by Sanford et al (1992) and later marketed by BIO-RAD Company.
A. Micro-carrier preparation
1. In a 1.5ml microfuge tube, weigh out 3 mg of microparticles (gold particles 1.0 mm) for 6 bombardments.
2. Add 1ml of 70 % ethanol, fresh prepared.
3. Vortex on a platform vortexer for 2-3 minutes.
4. Incubate for 15 minutes.
5. Pellete the microparticles by spinning for 5 seconds in a microfuge.
6. Remove the liquid and discard.
7. Repeat the following steps three times.
a. Add 1ml of sterile water.
b. Vortex for 1 minute.
c. Allow the particle to settle for 1 minute.
d. Pellete the microparticles by spinning for 2 seconds in a microfuge.
e. Remove the liquid and discard.
8. Add 50 ml of sterile water to bring the microparticle concentration to 60 mg/ml.
9. Store the microparticles at room temperature for up to 2 weeks.
B. Coating DNA onto microparticles
The following procedure is sufficient for six bombardments; if less bombardment is needed, prepare enough microcarriers for three bombardments by reducing all volumes by one-half. When removing aliquots of microcarriers, it is important to vortex the tube containing the microcarriers continuously in order to maximize uniform sampling.
1. Vortex the micrcarriers prepared in sterile water (60 mg/ml) for 5 minutes on a platform vortexer to resuspend and disrupt agglomerated particles.
2. Remove 50 ml (3 mg) of microcarriers to a 1.5 ml microfuge tube.
3. While vortexing vigorously, add in order:
a. 20ul spermidine (0.1 M)
b. 50 ul CaCl2 (2.5 M) was added drop by drop on vortex and continued for 2-3 minutes
4. Allow the microcarrier to settle for 1 minute.
5. Pellet the microcarriers by spinning for 2 seconds in a microfuge.
6. Remove the liquid and discard.
7. Add 140 ml of 70 % ethanol without disturbing the pellet.
8. Remove the liquid and discard.
9. Add 140 ml of 100 % ethanol without disturbing the pellet.
10. Remove the liquid and discard.
11. Add 48 ml of 100% ethanol.
12. Gently resuspend the pellet by tapping the side of the tube several times, and then by vortexing at low speed for 2-3 seconds.
13. Remove 6ul aliquots of microcarriers and transfer them to the center of a macrocarrier. An effort is made to remove equal amounts (500 mg) of microcarriers each time and to spread them evenly over the central 1 cm of the macrocarrier using the pipette tip and Desiccate immediately.
14. Fix macrocarrier, rupture disk and sieve on the instrument.
15. Build up 1100 psi pressure to the rupture disk and shoot the DNA on the cells of scueltellum callus.
16. Culture the tissue on the medium containing manitol (described above) to maintain the osmatic pressure.
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