Updates from November, 2012 Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 21:13 on 2012/11/25 Permalink

    I gave a quick look and the 4 long black worms that we collected in the last boat day look like the newest longissimus. I put them all in their new home. I think there is some platynereis for the menu this week.

  • Bruno Vellutini 22:46 on 2012/11/22 Permalink
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    Priapulus fertilization and RNA-seq time series 

    Fertilization of Priapulus brought from Kristineberg was successful. Sperm was added at 23h. I picked unfertilized +2000 oocytes into RNAlater and will continue to do this in the next 10d.

    Samples fixed: 0-7, 9d, 12d, 15d, 18d.

  • Bruno Vellutini 18:42 on 2012/11/20 Permalink
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    Picked 5 adult L. ruber and isolated each in small bowls in filtered uv-treated sea water. They will be used for genomic dna.

  • Bruno Vellutini 19:30 on 2012/11/16 Permalink
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    Lineus ruber and Membranipora in situ 

    Starting the first in situ in the nemertean Lineus ruber and trying different genes in Membranipora after establishing the ProtK timing. Selected genes are:

    L. ruber foxa six3/6 cdx gsc
    M. membranacea bra gsc piwi1 vasa

    All wells treated with 10 min in 1x protk concentration (0,01 mg/mL).


    Added probes.

    19 and 20/11/12

    Washing days, normal activity. Samples stayed in blocking buffer for 1h30 (instead of 1h) due to epic foosball match.


    Began developing at 11h. Twenty minutes later Mm bra cyphonautes shell were already completely blue… It seems that mucus cells in Lineus juveniles are being stained. No real signal coming up, except in six3/6, maybe. Mm piwi1 and vasa look like to be working, something is appearing. Exchanged AP once and let it at 4°C.


    Changed the AP of Lineus twice. Some signal kind of visible below the epidermis, nothing in the embryos. Stopped Membranipora development.


    Patterns in Lineus more visible and apparently real signal. foxa expressed in the gut region, six3/6 in the brain/anterior, cdx in the posterior tip (not in the epidermis), and gsc in the anterior mouth region. Let developing one more day at 4°C.


    Stopped reaction. Background began to come up, but I could check the expression more clearly. For example, foxa is detected in the mouth region and cdx in the anus. Now what needs to be done is remove the mucus cells with cystein before fixation.


    Restart development of Lineus to see if the signal get stronger in the juveniles and something appears in the embryos.


    Finally stopped the development. Will do ethanol washes tomorrow.

  • Bruno Vellutini 19:27 on 2012/11/13 Permalink
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    Probe synthesis for Lineus et al. 

    Started a probe pcr for synthesizing the following probes of Lineus ruber, Priapulus caudatus, and Terebratalia transversa:

    Species Gene ID Probe Kit
    Lineus ruber foxa BV196 SP6
    Lineus ruber six3/6 BV199 SP6
    Lineus ruber cdx BV204 SP6
    Lineus ruber gsc BV205 T7
    Priapulus caudatus tdr BV34 T7
    Priapulus caudatus mael BV35 SP6
    Priapulus caudatus gmcl BV183 T7
    Terebratalia transversa runx2 BV166 SP6


    Run gel.


    Dna extraction.


    Transcription reaction + precipitation.



    Lr foxa 688,23
    Lr six3/6 383,21
    Lr cdx 303,11
    Lr gsc 501,31
    Pc tdr 3853,39
    Pc mael 97,89
    Pc gmcl 2287,30
    Tt runx2 345,39

  • Bruno Vellutini 19:19 on 2012/11/13 Permalink
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    Took pictures of hedgehog and patched Terebratalia in situs. I think I covered the missing positions and stages.

  • Bruno Vellutini 13:35 on 2012/11/12 Permalink
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    Started organizing the expression patterns of Terebratalia for the segmentation project.

  • Bruno Vellutini 16:14 on 2012/11/05 Permalink
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    Some cloning of Lineus and Priapulus 

    Cloning of Pc gmcl, Lr caudal, Lr goosecoid, Lr foxq2, Lr fgf 8/17/18. Running the PCR with 60 °C for annealing, adjusted to primers Tm.


    Running gel:

    I cut caudal and goosecoid bands. Extracted, ligated/transformed.


    Colony PCR:


    Minipreps for cloning:

    Colony T7
    cdx 6 BV202
    cdx 7 BV203
    cdx 8 BV204
    gsc 6 BV205
    gsc 7 BV206
    gsc 8 BV207

  • Bruno Vellutini 14:27 on 2012/11/02 Permalink

    Fixed adult nemerteans 

    Because adult nemerteans are being kept in the lab with no use (they are starved and who knows if they will lay eggs in the next season) we decided to fix the adults for testing the in situ. I relaxed in 7.4% MgCl2, 5-10 is enough to get them completely relaxed, and fixed in 3.7% PFA for 4h at 4°C. Henrike did 3 washes of 10 min of PTw (last one overnight), plus 1 more PTw, mqH2O, and finally 100% MeOH. 16 worms were transferred to a 15mL falcon tube with methanol. Later this week I will fix more.

  • Bruno Vellutini 16:50 on 2012/11/01 Permalink
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    RNA Extraction of Priapulus staged samples 

    In order to estimate the amount of embryos needed for doing the RNA-Seq of specific stages I am extracting RNA from previousvly fixed samples. I extracted total RNA with Trizol, following Seaver protocol. Had to add equal volume of PBS to RNAlater to make embryos sink.

    Stage Embryos Volume (µL) ng/µL 260/280 260/230 RNA (ng)
    Gastrula ~650 20 80.22 1.55 1.16 1604.4
    Introvertula ~1070 20 88.89 1.78 1.04 1777.8
    2nd larva ~570 20 60.56 1.72 0.37 1211.2
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