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  • Bruno Vellutini 22:57 on 2015/05/11 Permalink
    Tags: , , pl10, ,   

    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: , , , , , , , , , pl10, ,   

    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 12:29 on 2012/07/23 Permalink
    Tags: , , , , , , pl10, , , , ,   

    PCR mostly stem/germ cell genes of Terebratalia 


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    Setting up a pcr with gradient (63 +- 3 °C) at 12h:

    Gene Primers Tm mean
    Mm Six3/6 64.10
    Tt Dicer1b 61.60
    Tt Runx 63.15
    Tt Mael 65.15
    Tt Piwia 65.55
    Tt Tudor1 61.95
    Tt Ago 62.95
    Tt PL10 63.10
    Tt Pum 65.35

    Extraction and ligation overnight at 4°C, except for MAEL.

    24/07/12

    Transformation and plating of colonies.

    25/07/12

    PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.

    27/07/12

    Put positive colonies to grow for minipreps.

    BV162 Terebratalia transversa Dicer1b
    BV163 Terebratalia transversa Dicer1b
    BV164 Terebratalia transversa Runx2
    BV165 Terebratalia transversa Runx2
    BV166 Terebratalia transversa Runx2
    BV167 Terebratalia transversa Tudor1
    BV168 Terebratalia transversa Argonaute
    BV169 Terebratalia transversa Argonaute
    BV170 Terebratalia transversa Argonaute
    BV171 Terebratalia transversa NK6
    BV172 Terebratalia transversa NK6
    BV173 Terebratalia transversa NK6
    BV174 Terebratalia transversa Wnt7
    BV175 Terebratalia transversa Wnt7
    BV176 Terebratalia transversa Wnt7

     
  • Bruno Vellutini 15:19 on 2012/05/24 Permalink
    Tags: , , , pl10, , , , ,   


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    Primers for more Terebratalia germline genes have arrived.

    • Tt dicer1b
    • Tt runx2
    • Tt maelstrom
    • Tt tudor1
    • Tt pl10
    • Tt ago
    • Tt pum
     
  • Bruno Vellutini 23:50 on 2012/04/01 Permalink
    Tags: , , , , , pl10,   

    More Membranipora genes: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b 


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    Set a PCR to clone: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b

    02/04/12

    Bands with expected lengths. Purified DNA, set ligation reaction for 1h45 at RT, heatshock transformation and plated colonies with Xgal.

    03/04/12

    Plates were wetty, so colonies did not spread well… some were forming strings; but enough to pick. Picked white colonies for colony PCR:

    09/04/12

    Sequencing PCR and dropped in the SeqLab.

    BV75 Membranipora membranacea Mex3 T7
    BV76 Membranipora membranacea Fez T7
    BV77 Membranipora membranacea Fez T7
    BV78 Membranipora membranacea Fez T7
    BV79 Membranipora membranacea PL10 T7
    BV80 Membranipora membranacea PL10 T7
    BV81 Membranipora membranacea PL10 T7
    BV82 Membranipora membranacea FoxG T7
    BV83 Membranipora membranacea FoxG T7
    BV84 Membranipora membranacea FoxG T7
    BV85 Membranipora membranacea Syt1a T7
    BV86 Membranipora membranacea Syt1a T7
    BV87 Membranipora membranacea Syt1a T7
    BV88 Membranipora membranacea Syt1b T7
    BV89 Membranipora membranacea Syt1b T7
    BV90 Membranipora membranacea Syt1b T7

     
  • Bruno Vellutini 11:56 on 2012/03/22 Permalink
    Tags: , , , , otp, pl10, ,   


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    Membranipora primers have arrived: Mm Syt1a, Syt1b, NK2.1, FoxG, Otp, PL10, Fez.

     
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