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  • Bruno Vellutini 11:41 on 2014/06/05 Permalink
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    RNA extraction of Lineus longissimus embryos and juveniles 


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    Starting extraction with snap-frozen samples of different stages of Lineus longissimus with the TriReagent protocol.

    # stage date ng/┬ÁL 260/280 260/230 total ng
    1 late cleavage 0d 02/04/14 204.0 1.89 1.83 4794.0
    2 blastula 1d 03/04/14 189.2 1.88 1.98 4446,2
    3 gastrula 2d 04/04/14 182.3 1.88 2.15 4284,05
    4 pilidium 3d 05/04/14 319.6 1.92 1.75 7510,6
    5 pilidium 5d 07/04/14 158.8 1.85 1.27 3731,8
    6 juvenile 2 months 12/05/14 50.3 1.77 1.03 1182,05
    7 juveniles 3 months 13/06/14 41

    Since the samples of the juveniles look so bad I think I’ll do another extraction.

     
  • Bruno Vellutini 11:26 on 2014/06/05 Permalink
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    Fixing Lineus longissimus juveniles 


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    Picked a batch of juveniles of the nemertean Lineus longissimus for in situ and immuno staining fixation.

    1. Relaxed for 1h in 7.4% MgCl2.
    2. Separated 1/4 of the worms for immuno on a tube.
    3. Just before adding the cystein I noticed that the worms were beginning to dissolve. So I only added 2% cystein in 7.4% MgCl2 for 30 s and immediately exchanged the solution for 4% FA. There are still some integer worms, so it should be ok. They should not be relaxed for more than 1h.
    4. Fixation for 1h at RT.
    5. Washed with PTw and then methanol for in situ samples.

    Relaxation seem to work, but many worms were not fully extended. The posterior end was curved ventrally.

     
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