Supplement to Microparticle Bombardment
By Omar Arnaout and Sudhir Nayak
o A (very useful) interactive user guide detailing the assembly of the hepta-adaptor and using the PDS-1000 system can be found at the bio-rad website (http://www.biorad.com).
- Alternatives to using the PDS-1000 hepta-adaptor
If you have a PDS-1000 but no hepta-adaptor you can just shoot the same plate 7-10 times and get similar results.
If you are unable to get your hands on a PDS-1000/He at all you can still do this!!! Other gene guns, including hand held ones like the Helios, are likely to work pretty well. A very simple gene gun was designed by Mike Nonet and it works for this procedure with minor modifications. Extensive details can be found at: http://thalamus.wustl.edu/nonetlab/ResourcesF/genegun/Genegun.htm.
I have used this gun to get lines, integrations, and germ line expression. The only disadvantages are that variability is higher and that is that it takes a longer time to shoot when compared to the PDS-1000 with the hepta adaptor.
- Egg plates generation protocol:
NOTE: Egg powder plates are not absolutely necessary. The same protocol can be used to grow large amount of unc-119(ed3) worms using NGM plates seeded heavily with OP50. However, if you want the plates to support the same number of animals you will have to use a 10x concentrated overnight culture of OP50 (details are below).
1) Prepare overnight OP50 LB culture
2) Prepare 30% (w/v) egg powder
In sterile water, vortex vigorously, use immediately. We get our egg powder from the local outdoor/camping store (REI). The brand name is Wakefield and the distributor is M. G. Waldbaum which is a subsidiary of Michael Foods (http://www.michaelfoods.com/).
We have also tested other brands of whole egg powder with some success. Honeyville: More contamination than others but it did work OK. This link to their direct site allows you to order 2.5lbs at a time.
Walton Feed: The egg powder from Walton feed does not work possibly because it contains dry milk among other things. I only tried it
once and plates were disgusting after 4 days and the agar started getting soft for some reason. They make other types but since other brands work better I did not follow it up.
The Bakers Catalogue: Never tried because its 2-4 times the cost of others.
The best bet is to try local stores that carry camping supplies. They will sometimes have 2-3 brands. I also found some at our local grocery store. NOTE: I’m currently using whole egg powder (not the yolk only powder). We have not tested yolk only powder.
3) Wash worms from 60mm NGM plate near starvation and pellet
4) Prepare 1:1 mixture of OP50 (15% egg powder final or 5x OP50 final)
5-15% egg powder depending on the brand and how much your worms like for optimal growth. Simply using more egg power does not always help and can actually decrease your yield. You can also use too much OP50 that will choke out your worms and reduce overall yield.
5) Resuspend worms in 0.5ml of 1:1: mixture of egg powder or 10x OP50.
6) Spread on 100mm NGM 100mg/ml nystatin plate
7) 10 x 100mm plates = 0.5-2ml packed worms after sucrose float using egg plates or 10x OP50 plates.
- Concentrated OP50 Plates (Alternative to egg plates):
If you plan on using concentrated OP50 instead the principle is the same. You need 10-20 60mm plates near starvation to start.
Grow 50ml of OP50 overnight, spin to pellet (For seeding 10 large plates)
Remove supernatant, add 5ml LB (PBS or M9 work too) and vortex well to resuspend. This is a 10X concentrated OP50 culture.
Wash worms from 10-20 60mm NGM plates (small plates) near starvation and pellet.
Add 10X concentrated OP50 to worm pellet, resuspend gently, and spread over 10 large plates (about 500ul/plate) just like you would seed plates with OP50. If you do this quickly the worms will not settle too badly and will be spread evenly over the large plates.
We have tried using terrific broth and other super rich media for growing OP50 but for reasons I don’t understand we get fewer lines. For this reason we use only L-broth. Similar to the egg plates, fresh OP50 seems to work better than older OP50.
- Sucrose Floatation Protocol:
60% w/v sucrose may be made ahead of time and kept at 4C.
Make sure 60% sucrose is chilled in ice-bucket for at least 15 minutes before using, break tip off a Pasteur pipette and flame to smooth edges (to be used for removing worms from sucrose)
Wash worms off of 100mm plates, and let settle for 10-15 minutes (or spin down) (fresh unwashed worms)
Resuspend in a small volume of M9 (4-5ml)
Mix with equal volumeof chilled 60% sucrose
Spin at 2000 rpm for 2minutes in Sorval RT6000
Bacterial debris willpellet, worms will float on the top of the sucrose (Worms floating on sucrose)
Remove worms quicklyusing a Pasteur pipette
Wash 2X with M9 toremove any residual sucrose
Note: There is usually a decrease in the number of worms available after a sucrose flotation, use only if necessary, and make sure that there are enough worms initially. Even very dirty plates can be used for shooting because moderately clean worms can be isolated using sucrose floatation.
The hardest part of his procedure is growing the unc-119(ed3) in large enough quantities so that you can shoot.
There are many pitfalls that can reduce the yield of worms and some issues are detailed below.
Transfers should be performed with sterile plugged pipets and sterile reagents. A laminar flow hood can also be used if severe contamination problems persist. You can also heat the egg powder mix to 60C for 1 hour if it happens to be the source of your troubles.
100 micrograms /ml nystatin (mycostatin) prevents most fungal growth. 500 micrograms /ml can also be used if fungal problems persist.
Low levels of contamination do not affect transformation. 1-2 additional rounds of sucrose floatation can be performed to clean up worms. However, multiple round of sucrose floatation will greatly reduce worm yield. Sucrose floatation is not always necessary or desirable so only use it if you have to.
Some brands of egg powder resulted in my plates getting very soft and disgusting. You can switch to a new brand of egg power or switch to using 10x concentrated OP50.
Seed one 60mm plate with 30-50 unc-119(ed3)(ed3) animals. 4-6 days later wash worms from plate near starvation and spread to 5-10 x 60mm plates. Each 60mm plate should have approximately 1000 worms near starvation.
100mm egg plates
Multiple 60mm dishes can be used to seed one 100mm plate. We frequently use 2 and up to 5 60mm plates for one 100mm
Egg powder must be vortexed vigorously and used immediately. Fresh cultures of OP50 work better than older cultures. The optimal concentration of egg powder varies with brand and ranges from 5-15%. Excessive use of egg powder reduces worm yield and makes purification of the worms difficult. If you are unable to find egg powder or the brands you find don’t work well then 10x concentrated overnight culture of OP50 also works but requires more planning.