I am trying to map a mutation using the method described by Wicks et al (2001). I have a drug resistant mutant strain that I crossed to Hawaiian males (CB4856). The resulting F1s I plated onto drug plates. I picked F2 worms that were able to
grow on the drug individually into pcr tubes containing 10uL lysis buffer, and stored them at -80C. In total, I picked 102 F2 worms.
I also picked the same number of wt N2 and Hawaiian worms, in equal proportion (51 N2 and 51 Hawaiian) in 102 different tubes for the control.
Next, I did proteinase K lysis to obtain 102 mutant and 102 control lysates. My question is, how do I form the bulk lysate of the 102 worms in one single, tube for the next steps of mapping (pcr and restriction digestion)? If I take 1uL of the
lysate from each of 102 tubes to make a final volume of 102uL of bulk worm lysate, then the DNA would get too diluted for the pcr step…is there a trick that I am
missing?
overall conc. of DNA in your bulk lysate is the same as the average of your individual lysates, but gives you an “average” genotype. so, you’re doing it right….
Yeah, I don’t really see the problem here - except that I’ve got no idea why you picked so many tubes of N2 and CB4856 controls. One of each (or maybe a bit more for volume) would have been plenty.
I guess I would say though that in my experience and according to what I’ve heard the crude lysate usually isn’t reliably stable, even at -80C. So I’m a bit concerned that you’ve made all these lysates ahead of time.
lysates definitely go bad. might as well pool the entire volume of all 102 mutant lysates then phenol extract and EtOH ppt (maybe add some glycogen carrier).
The point about having each lysate averaging out is right…thanks for that insight. I am debating if I should do the phenol extraction, or any handling with the lysate.
Maybe once the bulk lysate is formed it would remain pretty stable at -20C for some time. I have talked to a few people in the meantime, they also suggested single worm lysates are usually not very stable but that the bulk lysate stays fairly stable for a couple of weeks at -20C.