The principle of
transformation with a particle gun is a direct mechanical transport of the DNA
into the cell using metal beads as carriers. The general advantages of the
method are short handling time 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 Rapture 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. 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 vigorous 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. Suspend Au particles by
vortexing.
2. Prepare below 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. 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. Sterile micro-carriers
and stopping screens by soaking these ones in 70% ethanol for a few min.
2. Spot 10 µl of Au
particles coated with plasmid DNA on a micro-carrier.
3. Turn on power.
4. Load sterile rupture disk
into retaining cap and secure it to end of gas acceleration tube, tighten with
torque wrench.
5. Load macrocarrier and
stopping screen into microcarrier launch assembly.
6. Place microcarrier launch
assembly and target cells in the chamber and close door. (Distance = 6 cm).
7. Apply vacuum, when 28
inches of Hg are reached (it takes about 30 s), press quickly the vacuum
control switch through the middle VENT position to the HOLD position.
8. Bombardment: press FIRE
button until rupture disk bursts and helium pressure gauge drops to zero, then
release button.
9. Release vacuum by setting
the switch in 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 at least incubation of 12 hours.