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  • Bruno Vellutini 11:13 on 2015/05/12 Permalink
    Tags: , , , , wnt1   

    Novocrania wnt1 in situ with longer probe 


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    Na wnt1 Tt runx

    Did everything as usual and embryos looked normal.

    13/05/15

    Embryos quite damaged, including Terebratalia. Possible that pipette is not well calibrated?

    Restarted in situ today with extreme gentleness. Added 5 µL protk in 10 mL (instead of 2.5 in 5 µL) using the P20 pipette. Fosfatase wash with 83 °C and added hybe buffer around 1800.

    14/05/15

    Add probe.

     
  • Bruno Vellutini 22:57 on 2015/05/11 Permalink
    Tags: , , , , wnt1   

    Long probe for Novocrania wnt1 


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    Set probe PCR for:

    Na wnt1 BV387: T7 80 ng/µL
    Mm pl10 BV79: SP6 702 ng/µL

    and extracted.

    12/05/15

    Set transcription reaction and precipitation.

    13/05/15

    Finished probes (see values above) and made 1x dilution for wnt1.

     
  • Bruno Vellutini 18:24 on 2015/05/08 Permalink
    Tags: , , , , , , , , , , , wnt1   

    Membranipora in situ and Novocrania wnt1 


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    Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.

    wnt1 dlx bmp2/4 chordin fgf
    nanos vasa piwi1 piwi2 pl10
    Na wnt1

    Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.

    09/05/15

    Added probes just after synthesis.

    11/05/15

    Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.

    12/05/15

    Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.

    13/05/15

    Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.

     
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