Merbarone and camptothecin

Model Organism: Worms Scientific Discussion
1

Hi everyone–

I am trying to do some experiments where I soak L1s in either merbarone or camptothecin. Both are soluble in DMSO. The problem is that both also precipitate when I add them to 1X M9 solution. There are examples of both being used extensively in the literature, but details are sparse. I’m hoping someone has some experience using either of these chemicals and can give me some advice on their use.

Thanks!

Ash

2

Hi Ash,

I used camptothecin extensively during my PhD, although I should note that all of my experiments were done using either young adults or gravid adults… I looked back at my notes and here is the protocol I followed:

  1. Make 10mg/ml stock fresh for each assay (200-500μl should be fine) Heat for pulses in 100C thermoblock followed by vortexing to get it in solution (or can do longer heating in 55C waterbath)

  2. Make 10x working stocks for desired concentration:
    Conc ul Camp ul DMSO ul M9
    0nM 0 30 570
    1:1000 dil of stock 50nM 10.72 19.28 570
    1:1000 dil of stock 100nM 21.44 8.56 570
    1:10 dil of stock 500nM 1.072 28.93 570
    1:10 dil of stock 1000nM 2.144 27.85 570
    1:10 dil of stock 5000nM 10.72 19.21 570

Solution becomes a bit cloudy when you add the camptothecin. Concentration refers to final concentration, taking into account the 1:10 dilution performed by adding 200 μl of DMSO or camptothecin:DMSO solution in M9 to 2 ml of worm-bacteria mix in 6 well plates. I usually use really concentrated HB101 as food in the assay, putting approx. 50 µl of sludgy bacteria into the 6-well plate.

This protocol worked very well for me (Ward et al., 2007 EMBO J). I got a good killing curve with homologous recombination mutants and the assay was very reproducible in my hands. I also saw RAD-51 foci, a marker of DNA damage, reliably. Hopefully this assay can be adapted for L1s.

Good luck,

Jordan