From pamwayi from icipe.org Fri Jun 12 05:56:33 2009 From: pamwayi from icipe.org (Amwayi, Peris) Date: Fri Jun 12 07:30:29 2009 Subject: [Celegans] Native protein extraction Message-ID: Hi Dr. Aurelio, Greetings. I have just seen your request for assistance in native protein extraction. Am in that dilemma too, so just wondering if you got a solution yet. I want to do enzyme assay in locusts. Thanks and kind regards, Peris From ikklejaneo from yahoo.co.uk Mon Jun 15 15:42:04 2009 From: ikklejaneo from yahoo.co.uk (Jane Whittingham) Date: Mon Jun 15 17:33:03 2009 Subject: [Celegans] Microinjection frustration Message-ID: <213261.65125.qm@web23704.mail.ird.yahoo.com> I am desparately in need of some help with c. elegans microinjection. I am new to the worm field and have been learning to microinject for about 3 months now and have had absolutely no success whatsoever. I have tried asking other people in the lab to help out and have tried troubleshooting myself from sources on the net or from books on microinjection but nothing has helped. I seem to be doing ok injecting the worms in the right place from what I can tell, my problem is the recovery of the worms. I use M9 buffer for recovery and they initially seem to recover really well, thrashing about in the M9, but once the M9 is absorbed into the plate and the worm adheres to the bacterial lawn, the head is still moving around a lot, but the back end of the worm seems almost paralysed and after a day or so the worm dies. So far the main advice I have had seems to be that there are two main reasons for the worm not recovering: the cuticle is irreparably damaged during injection or the worm has dried out too much. I have tried to increase my speed of injection, I only inject one worm at a time and having timed myself typically take between 2-3 minutes to inject. I have tried tons of different thicknesses of agarose pad but nothing helped (I use 2% agarose in water). As for the damage to the worm, sometimes I can see this is a problem as the worm explodes after injection, but most of the time they seem absolutely fine, I have tried hundreds of different sizes and lengths of needles, tried breaking the needle straight on and at an angle, turned the worm at an angle to the needle so that only the smallest amount of the needle enters the gonad, but NOTHING has worked. Has anyone ever noticed this kind of thing happening before, I could really use some advice. Thanks so much! From khedges1 from yahoo.com Tue Jun 16 14:35:53 2009 From: khedges1 from yahoo.com (Kathryn Hedges) Date: Tue Jun 16 15:32:27 2009 Subject: [Celegans] Re: Microinjection frustration (Jane Whittingham) Message-ID: <275260.88329.qm@web82305.mail.mud.yahoo.com> > Date: Mon, 15 Jun 2009 20:42:04 +0000 (GMT) > From: Jane Whittingham > Subject: [Celegans] Microinjection frustration > To: celegans@magpie.bio.indiana.edu > Message-ID: <213261.65125.qm@web23704.mail.ird.yahoo.com> > Content-Type: text/plain; charset=iso-8859-1 > > I am desparately in need of some help with c. elegans > microinjection.? ... from what I > can tell, my problem is the recovery of the worms.? I > use M9 buffer for recovery and they initially seem to > recover really well, thrashing about in the M9, but once the > M9 is absorbed into the plate and the worm adheres to the > bacterial lawn, the head is still moving around a lot, but > the back end of the worm seems almost paralysed and after a > day or so the worm dies.? Jane, I forwarded your message to our local microinjection expert (Jacob Varkey) and he suggested a different buffer than M9: "Even though it is not necessary, in some cases, I have asked students to use a recovery buffer. There are different formulations of it. One is given below. 3mM HEPES (pH7.2), 3 mM CaCl2, 66mM NaCl, 2.4mM KCl, 4% glucose, 0.1% salmon sperm DNA. Some people simply add 4% glucose to M9. In either case, incubating the worms at 16 C for couple of hrs in recovery buffer (in a moist chamber) before transferring to a seeded plate is recommended." J.Varkey