I am trying to make males for crosses and have had trouble with heat shock protocols I have found online. I put 6 L4s on 5 plates and shocked them at 30 degrees for 6 hours. I used relatively late-stage L4s because I heard that was a good thing to do.
Problem is I got no males. I checked a few times after they started laying, and I even waited until there were a good amount of adults on the plate and there were no adult males.
Has anyone had success with heat-shocking to generate males? Am I doing anything wrong?
Sometimes it doesn’t work for me either. A colleague suggested 31degrees gave a better yield. However, now I just put late L4 worms in 7% ethanol for 30 mins, and a few days later you start to get males.
I usually heat shock way more than 6 L4s to get males. I usually do this on the order of 50-100 L4s and hope for the best. As for the EtOH, I have no idea, but came across this protocol from the Yanowitz lab website called “drunken hermaphrodites” a while ago, but have never tried it I must say. In it she gives the following reference: Protocol from Ralph Hecht (WBG 14(5):52 Feb 1, 1997)
I was shown a more convenient method to heat-shock for males: instead of putting plates in a 30 degree incubator for six hours you should put perhaps 50 or 60 late L4s on one seeded plate, wrap it with parafilm, and put it (weighed down) in a 33 degree water bath for one hour. After the hour, let the worms recover for a half-hour or more on your benchtop, then pick them over five worms apiece to each of six or more seeded plates. For most strains each plate will give you a few (zero to six, say) males in the next generation. It’s probably similar to the 30-degree-incubator method for effectiveness in making males, but I’ve found it’s much easier to forget the worms in the incubator over the course of six hours than to forget them in the water bath in one hour.
Still, for all that I recommend the above as being a more convenient method to induce X nondisjunction and get males, and for that matter the ethanol method might also be a more convenient method, suggesting either doesn’t really get at the question of why the method you already were using wasn’t working; it really should have been. The obvious possibilities are that your incubator isn’t really at 30 degrees, which is easy to check, or that your strain might be resistant to induction of X nondisjunction by heat shock or might be Her or Xol. I’d thaw (or request) a new copy of the wild-type strain N2, and see if you get better results inducing males with that. You might also make sure your strain is free of contamination; I don’t know of any report that contamination can affect the ability to heat-shock for males, but it’s not inconceivable, and keeping your strains clean is always good practice in any case.
I have always had good luck with Leon Avery’s old protocol - here’s what he has to say:
Heat shock is a very touchy protocol. You have to get the time exactly right: half an hour too long, and the worms are sterile, half
an hour too short, and there’s no nondisjunction. Furthermore, the correct time depends very sensitively on the temperature, and you
seldom know the temperature accurately enough. So I do as follows:
Put 5 L4 hermaphrodites on each of 6 plates at 30° C.
Remove 2 plates after 5.5 hours, 2 more after 6 hours, and remove last 2 plates after 6.5 hours.
One of those pairs should work. You’ll have advance warning which, because you’ll usually see some vulval abnormalities in the
parents, and they will produce small broods. If all your plates are sterile or if all are normal, adjust your incubator accordingly. The
frequency of males is still not high – you have to hunt for them.