SJ-500 PORTABLE GENE GUN Operation Manual
Contents
1. 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 lug ul 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 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 ol 6u golden or 1 6u golden or tungsten tungsten remark Fuda University Institute of Biophysics Academy o2. SJ 500 PORTABLE GENE GUN Operation Manual SLIENI Z ISO9001 2008 IS013485 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 tradeO2 scientz com sheray scientz com Website www scientz com Wwww scientzbio com 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 AProhibit open the gene gun 6 Prepare cells C Install and operation 1 Connect tube Use two sp
3. d 40ul Spermidine 0 1M vortex 30 sec add 100u1 CaCl 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 300ul 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 700u1 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 300ul absolute ethanol not disrupt the deposition quiescence min and then discharge the suspends 6 add 120ul ethanol and vortex to suspend solution If the solution disperse uniformity pipette 10ul 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 8ug DNA 10ul 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 ge
4. ecial 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 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 Sh
5. f Military Sciences Notes check references for details Apparatus solutions and gas need be prepared before experiments 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 0m most animal cells 1 6 1 7um microorganism 0 6um C Spermidine 0 1M filtrated CaCl 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 lug ul 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
6. ne 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 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 CaCl 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
7. oot 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 E Encasement list 1 Portable Gene Gun l 2 magazine siliconlizated 50 3 magazine turn shelf l 4 special wrench 14 17 2 5 movement wrench 300 36 1 6 9V batteries 2 7 high pressure t
8. ube 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 1 6um 1 0um 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 1 0 1 6um and for plants are 1 0um 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 I minute 5 centrifugation discard the suspend 6 add Iml 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 100ul suspend solution into centrifugation tubes each tube enough for 12 guns According to the quantity of sample to determine the tube numbers needed 3 add 10 20u1 plasmid DNA 1ug ul into the tube vortex 30 sec and then ad
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