Making Egg Media
Autoclave a metal blender, or sterilize a glass blender by running it with some bleach and water, and then rinsing it with sterile water. Boil 25 mL of water per egg. Wash the outside of all eggs with ethanol. Crack the eggs open into a sterile beaker and discard the shells. Dump the contents of the eggs into the blender, add the boiling water, and run the blender. When the solution is homogenous, dump the contents of the blender into a sterile beaker and let the froth settle. Make 6 mL aliquots of the egg solution into 15 mL conical centrifuge tubes. Heat the egg solution for one hour in a 65 °C water bath. Cool the solution to room temperature, and store it at –80 °C.
Concentrated HB101 bacterial culture is prepared by growing an overnight culture in 2xYT medium. Pellet bacteria at 4000 rpm for 15 minutes. Discard supernatant. Re-suspend bacterial pellet in M9 buffer, 1 ml buffer per gram of bacteria. Store at 4 °C.
Prepare starter cultures of unc-119 worms on 10 cm NGM lite plates made, seeded with a thick HB101 lawn. Allow the worms to starve out the plates. Store starved worms at 20 °C for up to 2 weeks. Inspect for contamination before using these to start egg-plate cultures.
Seeding Egg Plates
To prepare enough worms to perform one bombardment wash worms from four starved (less than two weeks old) 10 cm NGM lite/HB101 seeded plates with M9 buffer spiked with 10 mg/mL gentamycin. Pellet worms by spinning the worms for 2 minutes at 1500 rpm. Remove the supernatant. Wash twice with regular M9, and remove the final supernatant. Mix 6 mL of egg medium with 1.0 mL of concentrated HB101 bacteria. Add the mixture to the worms and mix with a pipet. Spread 25% of the mixture onto each of four 15 cm NGM lite plates and spread the solution gently. Grow 3.5 - 4 days at 20 degrees so that most worms are gravid adults.
Large-Scale Bombardment Using Hepta Apparatus
Incubate one seeded 10 cm NGM plate per bombardment in a 37 °C incubator for a couple hours to dry them out so that they absorb liquid. After incubation, put the plates on ice to chill. To avoid bacteria in the ice contaminating the dish, separate the two with a sheet of Saran wrap.
Use a siliconized microcentrifuge tube to prepare gold particles. Put a siliconized microcentrifuge tube on a balance, and zero it. Weigh out 30 mg of gold particles per tube. Add 1 ml 70% ethanol. Vortex the tube for 5 minutes. Let the tube sit for 15 minutes. Spin the tube briefly. Discard the supernatant. Wash the particles three times with 1 ml of sterile water. When removing the wash liquid (i.e. water), touch the pipette tip to the side of the tube opposite the gold. When adding water, steady the pipette by placing one finger on top of the pipette. After removing the water from the third wash, resuspend the gold in 0.5 mL 50% sterile glycerol. The final concentration should be 60 mg/mL. The gold can be stored for 1-2 months at 4 °C or room temperature.
Check solution of 2.5M Ca Cl2 for undissolved precipitate. If the solution contains undissolved precipitate, warm it in a 37 °C water bath. Thaw 0.1M spermidine.
To coat the gold with DNA, use a siliconized tube. Mix 50 mL (8-10µg plasmid miniprep) DNA with 100 mL of well-suspended gold particle solution in 50% glycerol. Vortex the solution for at least 1 minute. Add 150 mL 2.5M Ca Cl2. Vortex the solution again for at least 1 minute. Add 60 mL spermidine. Vortex 3 to 5 minutes. Re-freeze the spermidine at -80 °C. Tap spin the solution (i.e. spin very very briefly using the “pulse” button on the centrifuge). Spinning the solution too long will make re-suspention difficult or impossible. After the tap spin, remove the supernatant. Add 300 mL of 70% ethanol. Vortex the solution very well. The DNA makes the gold very sticky. Tap spin the solution again. Remove the supernatant. Add 170 mL of 100% ethanol. Vortex 5 to 10 minutes.
Add some dryerite and a kimwipe to an empty 15 cm plate. With tweezers, dip seven macrocarriers per bombardment, one at a time, into 100% ethanol and lay them on the kimwipe in the plate to dry. It is best to keep the plates in a fume hood. Open the lid to let the macrocarriers dry.
Wash out the bombardment chamber with ethanol on kimwipes. Wash the target plate shelf in a similar manner and put it in the bottom slot of the bombardment chamber. Wash the metal parts of the macrocarrier holders with ethanol and kimwipes. Load the macrocarriers into the lid of the macrocarrier holder with tweezers. Wash the red pressing tool with ethanol and kimwipes. Press the macrocarriers into the lid (part 4) with the red pressing tool and twist the tool. Add 20 mL of DNA-coated gold particles to the center of each macrocarrier, only covering the central area of the macrocarriers which lie above the holes in the metal macrocarrier holder. Then, split up any remainder of the DNA-coated gold particles among the macrocarriers, again only covering the central areas. Let the gold solution dry on the macrocarriers in the hood.
Wash unc-119 mutant worms off egg plates with sterile M9 salts, which can be spiked with 100 mg/mL streptomycin. It is easiest to add the M9 to the dishes and let them soak for a while. A disposable scraper can be used to dislodge worms. Then, remove the worms from the plates with pipettes. Transfer the worms to 50 mL conical centrifuge tubes. Centrifuge the worms for 2 minutes at 1500 rpm. Pipette off supernatant into another 50 mL conical centrifuge tube. The supernatant should be saved in case one accidentally sucks off some of the worms with the pipette. Resuspend the worms in M9. Let the adult worms settle to the bottom of the M9 by gravity for 15-30 min. Then, remove the supernatant, again saving the supernatant for safety. Plate about 2.75 mL of the worms from the four egg plates onto one dry, ice-chilled HB101 plate for each bombardment. Let the plates sit about 30 minutes to let the plates absorb the liquid. A confluent layer of worms should coat the whole plate.
Before an actual bombardment, it is recommended to test fire the apparatus. To test fire the apparatus, wash the two parts of the pressure divider (parts 1 & 2) with ethanol and kimwipes, and screw them together. Dip a rupture disk into ethanol with tweezers and shake off the ethanol. Place the rupture disk into the pressure divider. Screw the pressure divider into the bombardment chamber. Close the door of the bombardment chamber. Open the helium and adjust the pressure to at least 200 psi higher than the amount needed to rupture the disk. We usually use 1350 psi rupture disks. Turn on the vacuum pump. Turn on power to the bombardment chamber. Place the second switch on vacuum, and hold it until the needle on the meter reaches 28. Then, quickly turn the second switch to hold. Press and hold down the fire button. Release once the rupture disk breaks. One should hear a shot. Turn the second switch to vent, and turn everything off. Remove the burst rupture disk.
Wash the two parts of the pressure divider with ethanol and kimwipes. Dip a rupture disk into ethanol with tweezers and dry. Place the rupture disk into the assembled pressure divider. Screw the pressure divider into the bombardment chamber. Place an autoclaved stopping screen onto the pegs of part 6 of the macrocarrier holder. Invert the piece of the macrocarrier holder containing the macrocarriers (part 4), and put it onto the pegs of the part of the macrocarrier that lies beneath the stopping screen (part 6). Place the apparatus onto the metal shelf piece (part 7), and rotate the lever to lock the shelf onto the macrocarrier apparatus. Insert the macrocarrier apparatus complex into the bombardment chamber on the second shelf slot down from the top. The complex should fit right below the pressure divider. Rotate the lever of the macrocarrier apparatus such that the holes of the complex align with the holes of the pressure divider. Tape down the worm dish for bombardment to the target plate shelf. Use two pieces of tape on opposite sides, rolled such that the sticky side faces outward, and put them under the dish. If tape is not used, the dish will jump. Close the door of the bombardment chamber.
To bombard worms, open the helium, and adjust the pressure to at least 200 psi higher than the amount needed to burst the rupture disk. Turn on the vacuum pump. Turn on power to the bombardment chamber. Place the second switch on vacuum, and hold it until the needle on the vacuum meter reaches 28 of Hg. Then, quickly turn the second switch to hold. Press and hold down the fire button. Release the fire button once the rupture disk breaks. One should hear a shot. Turn the second switch to vent, and turn everything off. Remove the worms. Wash the worms off plates with M9 and distribute them among twenty 10 cm HB101 plates. Remove and discard the burst rupture disk and macrocarriers. Place the used stopping screen in the dish labled “used stopping screens.” Wash everything down with ethanol and kimwipes.
When finished with all bombardments, press the fire button while the machine is on vent in order to release residual helium pressure from the apparatus. This is important!! Residual pressure can damage the machine!!
Grow bombarded worms at 25 degrees for 10-14 days. Single out non-Uncs to screen for integrated lines. We single out 2 worms per plate. Those that give all non-Unc progeny are homozygous integrants. We then re-single 12-20 worms from potential heterozygous integrants (3/4 non-Unc) to get additional lines.
- 165-2226 Hepta stopping screens 50 pk $206.00 - enough for 50 bombardments, we autoclave and re-use these. $4.12/ea
- 165-2263 1.0um gold microcarriers 0.25g $335.00 - 40 bombardments. $8.40/ea
- 165-2330 1350 psi rupture disks 100pk $89.00 - 100 bombardments. $0.89/ea
- 165-2335 Macrocarriers (500) $370.00 - 70 bombardments. $5.29/ea
Source for Cheaper Gold (2 micron), works as well as BioRad one micron in our hands.
- Manufactured by Degussa
- They call the product 10K M gold
- It must be manufactured per order and they have a minimum order of 1 ounce with a setup fee for manufacture of ~$1000
- We ordered one troy ounce which is ~31 g for a total including setup of
about $1,300 about 3 years ago.
- The salesperson we went through was John Marcolini @ 978-465-4195.
- The contact number at Degussa manufacturing was 908-226-2047.
- The order was apparently placed through:
dmc2 Metals Group
3900 South Clinton Ave
South Plainfield, NJ 07080
- The main numbers for the company are:
Worms sitting on ice in hood waiting for bombardment.
Vortexing the gold with DNA.
Dipping macrocarriers in ethanol before drying and adding gold to them.
adaptor with rupture disk inserted.
Screwing in the Hepta adaptor into the bombardment chamber.
Pressure settings on the He tank.
PDS-1000 all set up. Just add worms.
Macrocarrier assembly and dry gold, ready to insert below Hepta adaptor.
Drying macrocarriers in the hood.
Adding DNA coated gold to the macrocarriers.
Assembling macrocarrier holder.