To all helpful micro-injectors,
Dear Worm breeders:
Thank you for all of the helpful hints for purifying rol-6. We got
a wide
range of responses to our injection problem. Several responses suggested that
CsCl was the most reliable method of producing a clean plasmid prep. The
Qiagen
prep seemed to be the most widely used, with the caveat that success rates
varies quite a bit. Two responses suggested a LiCl precipitation. Susan Mango
sent a LiCl and PEG precipitation protocol from Dave Zarkower that she has good
success with. Kouichi Iwasaki wrote that he uses Mellos published method and
resuspends the DNA in plain water. He uses plain water for injection as well.
He reported having problems with bad water at one point.
We also received advice that we needed to inject the mix alone
without the
rol-6 and just to inject water, TE, etc. to ensure that it was indeed the
rol-6
prep at fault.
We had already tried many of the suggestions received and were
pretty sure that
it was our rol-6 prep at fault. So the question was which method produced the
cleanest prep. We had tried many different mini preps and did not have good
luck with any of them. On the advice of one anonymous person we tried the
Qiagen again. This prep by itself (200 ug/ml) yielded rollers from 6 animals
injected, but the needle clogged at every other injection. On the advice of
Ralf Baumeister we did a PEG precipitation from this Qiagen prep. (1/2 volume
of 30% PEG; 1.5 M NaCl added to the prep, 3 hrs on ice, and 15 min. of
centrifugation at 4oC then resuspended the pellet in TE.) After injecting 6
worms with (200ug/ml) rol-6 alone, we saw no needle clogging, and 2 worms
produced rollers. I then injected rol-6 with our construct. The needle
did not
clog once during the injection of 30 worms, with this PEG precipitated
solution.
Of those 30 worms 5 produced F1s.
Perhaps we have shaken the gremlins.... Thanks to all who replied.
Beth De Stasio
Department of Biology
Lawrence University
Appleton, WI 54911
