Abstract
This protocol describes the basic work-flow of expanding nematode culture under lab conditions, which serves for the subsequent preparation of RNA (microarray), protein (IP), and DNA/protein (ChIP).
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from work performed by members of the Kenyon lab, including PZ. PZ was supported by a postdoctoral fellowship from the Larry Hillblom Foundation.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
If you refer to the first step of RNAi bacteria prep --- culture a single colony O/N in 1L of LB/Carb 100ug per ml/ Tet 12.5ug per ml (never grow them longer than 14 hours) and then concentrate it by centrifugation. Then wash the RNAi bacteria pellet with LB/Carb/Tet once (it is OK to wash it with sterile H2O), and resuspend the pellet with LB/Carb/Tet (1L to 50ml, 20X concentrated). Store it at 4C and this reservoir will be used as food later. Be careful with potential contamination from bacteria or mold. I have read an article from Michael Petrascheck’s lab about preparing bacteria stock for lifespan assays (JoVE 2011 49 Solis & Petrascheck). They also included anti-fungal in their recipe.To prepare a 15-cm RNAi plate, you just need to seed ~1.0ml regular RNAi culture (not the concentrated stock). Once you have seeded the worms (less than 6,000 per plate, it’d better to use 2.5% or 3.0% agarose to reduce burrow), remember to feed them with bacteria every other day. Spread ~1.0ml or more concentrated stock onto the plate, and make sure that it will dry soon (you don’t want to add too much liquid onto the surface). I’d say 1 plate of worms is quite enough for qPCR.BTW, I didn’t use liquid worm culture --- I used solid agar plates instead (you need to be careful with contamination problem, and it’s better to have several extra plates as backup).
I am wondering whether your RNAi agar plates contain Tet? And to make sure there is no contamination problem, do you just try to be careful or do you use any anti-fungal stuff?
Our stock agar plates are NG/Carb/IPTG (no Tet at all). Some people in the lab prefer not to use plates that already contain IPTG, and they dilute the bacteria ~5X to 10X and then add IPTG to 1.0mM for induction (either in liquid culture, or mix IPTG and diluted bacteria and then seed on the plate). This RNAi method appears to work pretty well. In my case, I didn’t include the anti-fungal agent as I was not sure whether it would have any lifespan effects. So try to be careful with your bench and clean it up with 70% ethanol before and after use. Keep the plates closer to the burner. Always seed the plates a few days before you start the worm culture, which would allow you to identify contaminations (typically mold) on the plates. This should work just fine. If you happen to see sporadic mold contamination, you can chunk the agar away and fix the hole with fresh 2% agarose.