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  • Bruno Vellutini 11:13 on 2015/05/12 Permalink
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    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
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    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
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    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.

     
  • Bruno Vellutini 20:09 on 2015/05/06 Permalink
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    Probe PCR Membranipora final genes 


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    Probe PCR for bmp/24, chordin, fgf8 and piwi1.

    07/05/15

    Extracted probe PCR gel.

    2015-05-07 11.35.03

    08/05/15

    Bit weird the bmp2/4, but well. Made the transcription reaction for 6:30h and put probes to precipitate.

    09/05/15

    Finish probes.

     
  • Bruno Vellutini 20:07 on 2015/05/06 Permalink
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    Novocrania wnt1 one more time 


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    Set PCR with 60°C annealing, 1:45 elongation, 40 cycles and primers at 25mM.

    wnt1 1:10 cDNA wnt1 1:20 cDNA
    pax6 1:10 cDNA pax6 1:20 cDNA

    07/05/15

    2015-05-07 11.35.14

    It worked. Extracted, ligated, transformed and plated.

    08/05/15

    Colony PCR and 2 volonies were fine:

    2015-05-08 17.18.24

    Put these to grow under the IDs:

    T7
    Na wnt1 long BV386
    Na wnt1 long BV387

    09/05/15

    Extract miniprep and set sequencing reaction.

     
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