Re: Microinjection frustration (Jane Whittingham)

[Kathryn Hedges]

> Date: Mon, 15 Jun 2009 20:42:04 +0000 (GMT)
> From: Jane Whittingham <ikkle...@yahoo.co.uk>
> Subject: [Celegans] Microinjection frustration
> To: cele...@magpie.bio.indiana.edu
> Message-ID: <213261....@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