ACT-LABS gun system Instrukcja Obsługi

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)Helios Gene Gun SystemInstructionManualCatalog

BatteryOne battery is provided with the Helios Gene Gun System. Under normal use, it shouldprovide approximately 1,000 discharges. For maximum life, o

Section 3Product Description3.1 Packing ListThe Helios Gene Gun System (see Figures 3 and 4) is shipped with the following compo-nents. If items are m

Fig. 3. Major components used for sample delivery with the Helios Gene Gun.Fig. 4. Components of the Tubing Prep Station.3.2 Identification of System

Fig. 5. Components and controls on the Helios Gene Gun.Gene Gun Controls DescriptionCylinder Lock Controls movement of the barrel pin. The cylinder lo

Push Bar A metal bar that ratchets the cartridge holder from one position to the next when the Cylinder Advance Lever is pressed. Move this bar to the

Fig. 6. Components and controls on the Tubing Prep Station, fully assembled.Fig. 7. The Tubing Cutter.Cartridge Extractor Tool (see Figure 8)A 12-pron

Fig. 8. Cartridge Holder and Cartridge Extractor Tool.Section 4Setting up the Helios Gene Gun System4.1 Inserting the Battery into the Helios Gene Gun

Fig. 9. Battery compartment. The battery compartment is located at the base of the handle of the GeneGun next to the connection for the helium hose an

Attaching the Helios Gene Gun to the Helium RegulatorComponents neededHelium regulator attached to a helium cylinderHelium hose assemblyHelios Gene Gu

Assembly of the Tubing Prep Station and SyringesComponents neededTubing Prep Station, baseTubing Prep Station, tubing support cylinderTubing Prep Stat

Warranty and Regulatory NoticesWarranty StatementThis warranty may vary outside of the continental United States. Contact your local Bio-Radoffice for

4. Cut a 12–13" piece of syringe adapter tubing; attach one end of the tubing to the barb of a 1/8"barb to female Luer fitting; attach the f

4. The nitrogen regulator should be turned on and adjusted to the correct pressure prior to con-necting the nitrogen line to the Tubing Prep Station.

5.2 Preparation of System Components Prior to BombardmentCalculating the Amounts of Gold and Plasmid RequiredPrior to precipitating DNA onto the gold

Table 2. Microcarriers and DNA Required for Various MicrocarrierLoading Quantities (MLQ) and DNA Loading Ratios (DLR)1Calculated Particle Materials R

ProcedureTime considerations: preparation of the DNA/gold suspension requires approximately 30 min.Several samples may be prepared simultaneously with

Loading the DNA/Microcarrier Suspension into Gold-Coat Tubing Using theTubing Prep StationMaterialsSuppliedTubing Prep Station (see Section 4.3)Gold-C

6. Remove the Gold-Coat tubing from the Tubing Prep Station. Turn off the flow of nitro-gen to the Tubing Prep Station using the knob on the flowmeter

Procedure1. Examine the coated the Gold-Coat tubing to verify that the microcarriers are evenly dis-tributed over the length of the tubing. Ideally, t

Fig. 12. Positioning the cylinder lock and the push bar in preparation for loading a cartridge hold-er into the Gene Gun. The cylinder lock has been p

c. Pull back and hold the cylinder advance lever to retract the inner barrel sleeve into the gun barrel (Figure 13).d. Place the empty cartridge holde

Note: This equipment has been tested and found to comply with the limits for a Class Adigital device, pursuant to Part 15 of the FCC rules. These limi

Fig. 15. LED display of the Helios Gene Gun. Using a pressurized system, once the cartridge holder ofthe Gene Gun is correctly inserted and engaged in

Notes: (1) The firing trigger is functional only while the safety interlock switch is pushedin. The safety interlock switch activates the trigger butt

Fig. 17. Loading cartridges into the cartridge holder. The numbers located on the outer rim indicatessample number when delivered by the Gene Gun.DNA

2. Turn the regulator valve counterclockwise until both the high and low pressure gauges onthe helium regulator register 0 psi. Several increase/decre

7. Immediately prior to DNA delivery, aspirate the media from the dish.8. Hold the dish perpendicular to the spacer and touch the end of the plastic s

5. Incubate 30 min to 4 hr under the appropriate conditions.6. Prepare the Helios Gene Gun for operation as described in Section 5.3.7. Immediately pr

Section 7Optimization of Gene Gun Parameters7.1 OverviewThe flexibility of the particle delivery system allows fine-tuning of experimental param-eters

Fig. 20. Luciferase expression in mouse skin transfected in vivo using the Helios Gene Gun.Skin homogenates were assayed 24 hours post-transfection.As

Another variable parameter, DNA loading rate (DLR), is determined by varying the con-centration of DNA precipitated on to the gold particles. Table 3

Table 4. Suggested starting conditions for in vitrotransformationof tissue culture cells using the Helios Gene Gun.Parameter ConditionsHelium pressure

Table of ContentsPageSection 1 General Safety Information...11.1 Helios Gene Gun Sa

1. Rewash remaining microcarrier prep 2–3 times using a fresh bottle of 100% ethanol.2. Flush Gold-Coat tubing with nitrogen gas for 15 min prior to l

Possible solutions 1. Make sure DNA is resuspended in TE or distilled water, not saline.2. Check that DNA has been precipitated onto the microcarriers

(polyvinylpyrrolidone, MW 360,000), 0.5 g, Cartridge Collection/ Storage Vials, 5, Dessicant Pellets, 5, Gold-Coat Tubing, 50 ft.165-2432 Helios Gene

EnvironmentalOperating 50 °F ( 10 °C) to 90 °F (32 °C) temp.30–80 % humidityStorage 32 °F (0 °C) to 140 °F (60 °C) temp.10–90 % humidityTubing Prep St

2. To measured gold, add aqueous solution of mRNA preparation.The ratios of RNA to gold used are similar to those used for DNA (1–15 µg RNA/mg particl

To replace the O-ring on the end of the barrel liner:1. Remove the barrel liner.2. Remove the old O-ring from the barrel liner.3. Place a new O-ring i

10.4 Replacing the razor blade on the Tubing Cutter and disas-sembly of the unitThe cutting edge of the Tubing Cutter is a standard, single edge razor

3. Raise the arm up and away from the base.4. The Tubing Cutter may be cleaned with soap and water and /or with 70% or 100% ethanol.Do not autoclave t

Discharge into ParafilmMaterialsParafilm laboratory film (American Can Company)Glass plateProcedure1. Cut a piece of Parafilm from the roll; a 1"

Fig. 26. Representative cross-section showing penetration of gold particles into water agar afterdischarge with a helium pulse.10.7 Quantitation of DN

Section 1General Safety InformationCaution: In particle bombardment DNA-coated microparticles are accelerated to velocities in excess of 1,000 ft/sec

10.8 References1. Albertini, M. R., Emler, C. A., Schell, K., Tans, K. J., King, D. M. and Sheeby, M. J., Cancer GeneTher., 3, In press (1996).2. Andr

10.9 Quick Guide to OperationBefore the Bombardment1. Coat microcarriers with DNA, load into tubes, and prepare cartridges prior to day of exper-iment

Life ScienceGroupWebsite www.bio-rad.com Bio-Rad Laboratories Main Office 2000 Alfred Nobel Drive, Hercules, CA 94547, Ph. (510) 741-1000, Fx. (510)7

Fig. 1. Location of the instrument serial number label on the Helios Gene Gun.1.4 Ear and Eye ProtectionCaution: Expansion of gas from high pressure t

Table 1. Advantages of particle bombardment forin vitroand in vivogene transfer.• Easy to use, rapid, versatile gene delivery system• Independent of t

Prior to transfection, the plasmid DNA must be attached to the gold particles. This is accom-plished by precipitation of the DNA from solution in the

Preparation of the gold/DNA tubes used in the Gene Gun requires an area approximate-ly 1 m2for the Tubing Prep Station, for manipulating the tubing, p