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  • Bruno Vellutini 18:00 on 2012/04/30 Permalink
    Tags: debug,   

    Debugging contamination of Wnts 

    Four of the sequences from 2012-04-25_0446 were contamination of Terebratalia Wnts in my samples. Wnts had already appeared in the sequences of Henrike, but the source was not identified. To identify if the contamination is in the sequencing reaction or the actual Wnts are being cloned Chema suggested to run a PCR with the miniprep using the gene specific primers. I also included the specific Terebratalia primers to see if there is any amplification.

    ID Gene Actual sequence
    BV93 Mm Piwi1 Tt Wnt16
    BV95 Mm Piwi1 Tt Wnt10
    BV99 Tt Nanos Tt Wnt10
    BV104 Tt Nanos Tt Wnt16

    Debug PCR for Wnt contamination

    The PCR shows that the actual Wnts were being cloned. LB medium used for transforming or growing should be the culprit.

     
  • Bruno Vellutini 18:00 on 2012/04/30 Permalink
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    Probe synthesis for Mm Vasa, Nanos, Piwi1, Piwi2 and Tt Vasa, Nanos, Piwib 

    Running a probe PCR for germ line genes of Membranipora (again) and Terebratalia (see table below). Setup is the same as a colony PCR, but doubling the amount (50µL) and using 3 µL of plasmid DNA. I extracted the bands and purified the DNA.

    Membranipora probe pcr for nanos, piwi2, vasa, and piwi1Terebratalia probe pcr for vasa, nanos, and piwib

    01/05/12

    This is the gel with the purified yield and the nanodrop measures.
    Purified dna from probe pcr of Mm and Tt germ genes

    02/05/12

    Transcription reaction initiated at 11h, tubes put in the cycler using incubate function at 37 °C.

    03/05/12

    Finished the probe synthesis and stored in hybe buffer diluted 50 ng/µL.

    Probe specs
    ID Gene Probe Kit Probe PCR ng/µL  Probe ng/µL
    BV3 MmNanos SP6 881.0 1283.41
    BV6 MmPiwi2 SP6 745.1 1149.77
    BV91 MmVasa SP6 586.9 477.97
    BV94 MmPiwi1 SP6 571.3 902.3
    BV96 TtVasa SP6 517.8 572.53
    BV100 TtNanos SP6 582.4 1158.87
    BV103 TtPiwib SP6 651.8 493.36

     
  • Bruno Vellutini 12:00 on 2012/04/29 Permalink
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    Abstract submitted to EuroEvoDevo 2012 

    Germ cell development in non-spiralian lophotrochozoans: insights from a bryozoan and a brachiopod

    Bryozoa and Brachiopoda are two spiralian taxa that, unlike other spiralians, undergo non-spiral cleavage and have unique non-trochophore larvae. Previous morphological studies determined that no distinctive germline is formed during embryonic development and that germ cells first appear relatively late in larval life or after metamorphosis. These observations suggest that the specification of primordial germ cells occurs by late inductive signaling (epigenesis) rather than inheritance of maternal determinants (preformation). The molecular mechanisms involved in germ cell formation in bryozoans and brachiopods are currently unknown. We have therefore used RNASeq data to identify and then clone the conserved germline-specific genes vasa, nanos, and piwi from the bryozoan Membranipora membranacea and the brachiopod Terebratalia transversa. In situ hybridization shows that Mm-nanos transcripts are not detected in the blastomeres during early cleavage, but are localized posteriorly in the internal sac region of the late-gastrula stage and early cyphonautes larvae of M. membranacea. In addition, Mm-piwi2 mRNA is present in the cytoplasm of M. membranacea blastomeres and is broadly expressed in the larval tissues, except for the corona and apical organ. Our preliminary results suggest that the signaling related to the differentiation of bryozoan germ cells may be established earlier in ontogeny than previously thought, possibly during gastrulation. A thorough analysis of the expression patterns will provide clues for understanding the regulatory mechanisms of pluripotent and germ cell development in bryozoans and brachiopods and offer further insights about the developmental diversity of spiralians.

     
  • Bruno Vellutini 17:20 on 2012/04/27 Permalink
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    Sequencing for Terebratalia segmentation genes cloned by Katrine/Andi. Just ran a sequencing PCR and sent the products to the sequencing facility.

    03/05/12

    Quickly checked the sequences today and they look bad. The only genes properly cloned were Patched and Netrin. The single Netrin sequence looks ok while the 2 Patched ones are a bit trashed.

     
  • Bruno Vellutini 18:00 on 2012/04/26 Permalink
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    First try on the confocal with L. ruber. Samples treated cleared with Murray’s clear and stained with Phallacidin and Propidium Iodide. Since I only used the 40x objective I just managed to capture close ups of the body wall and anterior end. I could only do one slide using the control with murray clear in the confocal, so I will still compare with the samples without murray clear.

    Tissues looked quite ok. By checking out in the scope with DIC the epidermis looked normal and the cilia were intact, it seems. Staining in both (cysteine/control) looked the same under fluorescent lamp.

     
  • Bruno Vellutini 12:30 on 2012/04/26 Permalink
    Tags: , , ,   

    Primers Tt en, Tt dlx, and Mm six3/6 just arrived.

     
  • Bruno Vellutini 18:14 on 2012/04/23 Permalink
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    Fixed 4 samples for confocal microscopy.

    • 10 days, jelly dissolved with 5 min (stirring) + 5 min (while dissecting) 5% cysteine.
    • 10 days without cysteine.
    • 21 days, jelly dissolved with 10 min 10% cysteine.
    • 21 days without cysteine.

    10% cysteine damaged the juveniles and some died. This concentration is too high, even with short times. 5% cysteine worked well with the earlier stages and it was much easier to dissect the eggs off the bundles afterwards (compared to the non-treated samples). The only thing is to see if the cysteine at this concentration damaged the embryos (or could affect the in situs).

    Relaxation with MgCl2 is not working so well, the trunk of the juveniles from the 21 days samples were completely retracted. Or maybe try for a longer time (Maslakova2009 waited 10-15 min until they were completely relaxed). Also, they use poly-l-lisine treated slides.

     
  • Bruno Vellutini 16:05 on 2012/04/23 Permalink
    Tags: , , , , ,   

    Colony PCR Membranipora Vasa and PiwiL1 and Terebratalia Vasa, Nanos, and Piwis 

    Colony PCR with the selected genes from the previous batch of heat shock transformations.

     
  • Bruno Vellutini 16:13 on 2012/04/22 Permalink
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    Introducing stirring to help liquify the jelly. Also trying with lower concentrations, because 5 min in 10% cysteine kill planarians.

    Try 5 min stirring of (check after every minute):

    • 10% cysteine + MgCl2
      • 15 min to loosely attached bundles.
    • 5% cysteine + MgCl2
      • 30 min to loosely attached bundles.
    • 10% cysteine
      • 10 min to loosely attached bundles.
      • 15 min to good liquifying ratio.
      • 20 min to complete dissolving of jelly.
     
  • Bruno Vellutini 18:10 on 2012/04/21 Permalink
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    Tried the consensus protocol for fixation from scratch. Consists of removing the outer case and immersing the bundles and jelly in Cysteine+MgCl2. Concentrations tried in a 35mm dish at 18 °C (no stirring):

    2mL 10% cysteine + 2mL MgCl2: Dropped the bundles and observed what happened to the jelly and how easy it was to remove it. It seems that the cysteine does liquify the jelly slightly, but after 15 min it was still there a somehow sticky.

    Since this gave no results I did not try lower concentrations and tried to use 10% cysteine directly (without MgCl2) which strangely did not give much results.

     
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