SJ-500 PORTABLE GENE GUN Operation Manual

SJ-500 PORTABLE GENE GUN Operation Manual
SJ-500 PORTABLE GENE GUN
Operation Manual
ISO9001: 2008
ISO13485--2003
NINGBO SCIENTZ BIOTECHNOLOGY CO., LTD
ADD:No.65 Mujin Road, National Hi-tech Park, Ningbo, China
TEL:0086-574 88352466 FAX: 0086-574-87113393
M.B: 0086-13967868094 Post Code:315013
E-mail: [email protected]
[email protected]
Website:www.scientz.com www.scientzbio.com
1
A. Principle and Structures
1. Principle: SJ-500 Portable Gene Gun is a gene transfer apparatus for directly
shooting DNA bullet. It is used helium as a power to carry DNA bullet to the body. It
is fitting to use in plant in vivo, plant tissue, and cultured cells.
2. Structures
B. Before experiment (Dry environment)
)
1. Prepare all the apparatus, materials and tools needed.
2. Unscrew the shoot tube and soaking in 75% ethanol for 15 minutes, and try.
Clean the gun with 75% ethanol and try. After cleaning the shoot tube, make sure to
screw it tightly. Clean the magazines base, bar nut, and bar net with 75% ethanol and try.
3. Clean the high-pressure tube. When use a new gene gun, first connect one end
of the tube with the helium steel bottle, let the air clean the tube for 1-2 seconds. Then
connect the other end of the tube with gene gun.
4. Prepare the sterilized solutions, tools and apparatus (such as ethanol, cleanse
desk, tips etc.)。
5. Prepare cover DNA bullets.
▲Prohibit open the gene
gun.
6. Prepare cells.
C. Install and operation
1. Connect tube: Use two special wrenches connect the tubes connecting the
gene gun and the helium steel bottle. Make sure it is airproof and no leak. Open the
valve of air steel bottle. Record the indicator value of high-pressure meter, and adjust
the pressure at 0.4-3.5Mpa to 5.0Mpa.
2. Install magazine Pull down the dustproof cover (Use thumb and forefinger to
catch the cover using). Use right hand to pull back rolling trigger to the end, and use
left hand to pull fore the lock pole and orientation to the right. Loose rolling trigger,
put the empty magazine to the base (notice the direction, concave is fore and
protruding is rear). The magazine tightly clings to the fore end of the base. Put in the
base and move back to 2mm. Press close to the back end, Lock pole reposition, and
Lock magazine. Use left hand to put down magazine and move right and left slightly
till a “click” sound. Cover the dustproof cover.
2
3. Try shoot: Press the safety button till the indicator light bright (finish charge),
then press shoot trigger. If first use, you can shoot empty to make sure the air and the
electricity are at normal situation. Pull out the empty magazine.
4. Set bar net: Screw the bar nut in the shoot tube end, and put in bar net slice.
And then screw the bar nut tightly.
5. Set DNA bullet: Use tip to transfer bullet to the cavity. Put in a culture dish
containing dryer. When it little dry, can be used.
6. Put the bullet cavity into magazine as step 2, and set dustproof.
7. Shoot: Hold gun, use thumb to press safety button. The indicator light will
light and then turn to red, put the shoot tube in the 0~2cm site of the receptor. For
living animals, can touch the skin. Mouse (belly or ear)、rat (belly, ear or thigh), dog
(nose or other sites), pig (nose or other sites). The hairs should be got rid of using
shaver or depilitant. Use 75% ethanol clears the skin. The press of helium is set at
2.5~3.5Mpa. If use tissues or cells, the press is set at 0.8~1.2Mpa. Press the trigger
and shoot.
8. Second shoot: Press rotation trigger, let magazine turn, and a “toot” sound
will be heard. Press trigger.
9. Repeat step 8 till the twelve shoots (Note: when the indicator light is off, need
press the safety button to recharge).
10. Pull the dustproof cover, and drop out the magazine. In case contamination,
suggest use bullet once.
11. For the second cycle, change new magazine.
D. After experiment management
1. Fasten up the high-pressure valve of steel bottle. Shoot with no bullet to let
remain air out. Pull gun, high-pressure tube, pressure meter, and valve etc.(according
to the use manual of steel bottle to maintain the steel bottle). If long time no use, pull
out the batteries.
2. Use 75% ethanol to clear the parts and put into box.
3
E. Encasement list
1. Portable Gene Gun
1
2. magazine (siliconlizated)
50
3. magazine turn shelf
1
4. special wrench(14*17)
2
5. movement wrench 300*36
1
6. 9V batteries
2
7. high-pressure tube
1
8. decompress valve
1
9. bar steel net
50
10. gland
10
11. special tool for gland
1
12. guarantee card
1
13. user manual
1
14. quality certification
1
Note: golden power φ1.6µm, φ1.0µm can be selected
For reference (confidential materials, only for the user)
)
(B)
)the preparing of DNA bullet
Step 1:
:Clear the golden powder
The golden powder used in animals are usually φ1.0~φ1.6µm, and for plants are
φ1.0µm.
(1) put 60mg golden powder into siliconlizated Eppendorf tube, add absolute
ethanol.. Use ultrasonic crusher them till hot temperature(about 3~5 minutes).
Transfer the golden powder into another siliconlizated Eppendorf tube.
(2) centrifugation at 4000~6000rpm, discard the suspend.
(3) add 1ml absolute ethanol, vortex 3~5 minutes. Transfer the golden powder
into clear siliconlizated Eppendorf tube.
(4) quiescence 1minute.
(5) centrifugation discard the suspend.
(6) add 1ml sterilized water, vortex, Transfer the golden powder into clear
siliconlizated Eppendorf tube. centrifugation discard the suspend. (repeat 3 times)
(7) put 1ml 50% glycerol into the precipitation.
Second Step: the preparing of DNA bullet.
(1) Vortex the golden powder in 50% to become suspended solution.
(2) pipette 100µl suspend solution into centrifugation tubes (each tube enough
4
for 12 guns; According to the quantity of sample to determine the tube numbers
needed).
(3) add 10-20µl plasmid DNA(1µg/µl)into the tube, vortex 30 sec., and then add
40µl Spermidine (0.1M), vortex 30 sec., add 100µl CaCl2(2.5M) while vortex, 30
sec. ~ 1 min., quiescence for 1min, then transfer the golden powder into a clear tube.
Centrifugation, and discharge the suspended solution.
(4) add 300µl 70% ethanol, mix with fingers (no need vortex). If the deposition
disperses uniformity, centrifugation, and discharge the suspended solution, add
absolute ethanol and then go to next step. If the deposition cannot be dispersed, the
ultrasonic need be used (add 70% ethanol till 700µl, 100~200W, 1 sec, intermission 1
sec, repeat
4~6 times,. Transfer the golden powder into a clear tube siliconlizated
Eppendorf tube. Centrifugation, and discharge the suspended solution.
(5) add 300µl absolute ethanol, not disrupt the deposition, quiescence 1 min, and
then discharge the suspends.
(6) add 120µl ethanol and vortex to suspend solution. If the solution disperse
uniformity, pipette 10µl into bullet (enough for 12 bullets). First put the bullets into
the mixer, run slowly (make the golden powder to the tube wall). Make sure the
suspend solution go to the bullet and not let the golden powder at the below 3 mm of
gas hole. When the solution in the hole is dry, bombardment can begin. The
bombardment starts from the control.
For each bullet, 0.5mg gold, 0.8µg DNA (10µl plasmid DNA). More plasmid
DNA may be better. Too high concentration of DNA may make the particle
congregate.
(C)
)The transformation mechanism of gene gun
The gene gun technology is a novel technology of delivering genetic materials
into tissues, cells and organelles with high speed.
SJ-500 Portable Gene Gun has a special apparatus to give rise to high-pressure
gas wave from 0.4Mpa to 5Mpa. This mechanism makes the DNA into the receptors
with “cold” gas and avoids damage cells by “hot” gas wave.
5
SJ-500 Portable Gene Gun can be used in plant in vivo and provide stable and
high-efficiency transformation.
(D)
)The Physical and Chemical factors of affecting the gene gun
transformation
The physical and chemical factors mean some parameters of gene gun such as
pressure, distance, bombardment times, the concentration and purification of DNA,
the concentration of DNA precipitator (CaCl2, Spermidine) etc. According to the
characteristics of receptors, these parameters may be adjusted.
(1) The pressure selected will affect the penetrability. If he receptors are different,
the pressure will different. As a result, according to the materials used to adjust the
pressure.
(2) The distance is another important factor. At the same power, longer distance
means lower penetrability. And shorter distance means bigger damage to the cells. As
a result, suitable distance should be considered.
(3) Experiments have proved that the bombardment times will affect the
transformation. Multiple bombardments will increase the instantaneous expression.
However, too many bombardments may damage the cells. For these reasons, 2 to 3
guns is suitable for one position.
(4) The higher purification of DNA, the higher efficiency of transformation.
However, the transformation efficiency is not consistence with the concentration of
DNA. Too high of DNA concentrations will make them to congestion and low the
efficiency. The most common used concentrations of DNA are 1µg/µl.
(5) The effect of DNA precipitator is very big. The suitable concentrations of
CaCl2 and spermidine are 1.9-2.5M (suggestion 2.5M) and 100mM, respectively.
★ Cautions: When operate gene gun, defend glasses and glove are needed.
No person is allowed to stand on the back of the target.
(E)
)Notes
6
(1) When the battery valve cannot be open and the indicator light cannot be
turned red, this means the power insufficiency and need change battery (9V).
(2) When gas pressure is low and gas leaks, screw the beam tube and check the
airproof ring. When change the rear ring, please use special tool.
(3) The prepare and store of DNA bullets should not be affected with damp. Put
dryer into the bullet box.
(4) The beam trigger does not work; the rotation trigger cannot be moved.
(F) Experiment conditions
Name of the receptor
mouse
rat
Shoot position
abdomen
Liver or kidney
Pressure
2.5Mpa
1-1.5 Mpa
Gas
Helium
Helium
Shoot Distance
cling
1-3mm
Metal Particle
φ1.6µ golden or
φ1.6µ golden or tungsten
tungsten
remark
Fuda University,
Institute of Biophysics
Academy of
Military Sciences
Notes: check references for details.
Apparatus, solutions and gas need be prepared before
experiments
7
1. The purity of helium or nitrogen is 99.999%. And their pressure should be
above 9Mpa, commonly 15 Mpa.
B. Golden or tungsten powder. (for plant cells, 1.0µm; most animal cells,
1.6~1.7µm; microorganism, 0.6µm).
C. Spermidine (0.1M, filtrated), CaCl2 (2.5M, filtrated), sealed 100% ethanol,
70% ethanol, 50% glycerol, and sterile distilled water.
D.
JY92-II
ultrasonic
crusher,
XW-80A
vortex,
table
high-speed
micro-centrifuge (12000 rpm) (Scientz provided).
E. Receptor(transformation materials)
,donator(plasmid DNA, 1µg/µl, about
10kb, had better no more than 15kb).
The conditions above should be prepared before open the gene gun box. What
kind of transformation materials or how to detect the instantaneous expression, please
consult the representatives.
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