The
principle of transformation with a particle gun is a direct mechanical
transport of DNA into cells using metal beads as carriers. The general
advantages of the method are short handling times and high efficiency, although only
transient transformants are obtained in our hands.
[Material]
E Bio-RAD
PDS-1000/He
E Au particle (1.6 µm diameter Au powder, Bio-RAD 1652264)
E Rupture
disk
E Stopping
screen
E Micro-carrier
[Solution required]
E 2.5 M CaCl2
(filtered)
E 0.1 M
Spermidine (filtered)
E 70%
ethanol
E Absolute
ethanol
E 50%
Glycerol (Autoclaved)
[Procedure]
1) Sterilizing of Au particle
1. Put 60 mg
of Au particle in a 1.5 ml microtube.
2. Add 1 ml
of 70% ethanol in the microtube.
3. Vortex
vigorously for 5 min.
4. Let it stand
for 15 min.
5. Centrifuge
at 15000 rpm (14000 x g) for 5 sec.
6. Discard
the supernatant.
7. Add 1 ml
of sterile water.
8. Vortex
vigorously for 2 min.
9. Discard
the supernatant.
10. Repeat
from step (5) to (9) twice.
11. Add 1 ml
of 50% glycerol and vortex vigorously.
12. Store at
–20˚C.
1. Resuspend
Au particles by vortexing.
2. Prepare the
following solution:
Au particle glycerol stock@@@@@@ 50 µl
Plasmid DNA@@@@@@@ @@@ 10 µg (0.2`1.0 µg/µl)
2.5 M CaCl2@@@@@@@@@@@ 50 µl
0.1 M Spermidine@@@@@@@ 20 µl
3. Vortex for
2 min.
4. Let it stand
for 15 min.
5. Centrifuge
at 15000 rpm (14000 x g) for 5 sec.
6. Discard
supernatant.
7. Add 150
µl of 70% ethanol and vortex.
8. Centrifuge
at 15000 rpm (14000 x g) for 5 sec.
9. Add 150
µl of 100% ethanol and vortex.
10. Centrifuge
at 15000 rpm (14000 x g) for 5 sec.
11. Discard
supernatant.
12. Add 50
µl of 100% ethanol and vortex.
1. Sterilise
microcarriers and stopping screens by soaking these in 70% ethanol for a few
minutes.
2. Spot 10
µl of Au particles coated with plasmid DNA on a microcarrier.
3. Turn on PDS-1000.
4. Load a sterile
rupture disk into retaining cap and attach it securely to the end of the gas acceleration
tube, tightening with a torque wrench.
5. Load a microcarrier
and a stopping screen into the microcarrier launch assembly.
6. Place the microcarrier
launch assembly and the target cells in the chamber and close the door.
(Distance = 6 cm).
7. Apply
vacuum. When 28 inches of Hg are reached (it takes about 30 s), quickly press
the vacuum control switch through the middle VENT position to the HOLD
position.
8. Bombardment:
press FIRE button until the rupture disk bursts and the helium pressure gauge
drops to zero, then press the release button.
9. Release vacuum
by setting the vacuum control switch to the VENT position.
10. Remove
plates, then the macrocarrier and stopping screen, then the rupture disk.
11.
Incubate the plates at 25˚C and observe after
incubation of at least 12 hours.