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  • Bruno Vellutini 18:44 on 2012/10/26 Permalink
    Tags: , , , synaptotagmin   

    Membranipora in situ Syt1a and Nanos 

    In situ with Membranipora for testing the newly fixed material with glutaraldehyde. I’m using 4 conditions and 2 genes:

    ProtK Time Gene Gene
    0.5x 10 min Syt1a Nanos
    0.5x 20 min Syt1a Nanos
    1x 20 min Syt1a Nanos
    2x 20 min Syt1a Nanos


    Developing the in situ. Wells with nanos already showing the background all around with no difference from treatments. Synaptotagmin looks more promising, no background still. First well seems to have a slight purpleish regions, but others are plain white. Exchanged the AP twice and let it overnight in cold room.


    Nanos is completely background. On syt1a I think there is signal in the last two treatments (1x and 2x). The lighter treatments seem to have some background. I exchanged the AP twice and will stop the reaction today.


    It seems that synaptotagmin works.

  • Bruno Vellutini 18:49 on 2012/10/12 Permalink
    Tags: , , synaptotagmin   

    Membranipora in situ with new material 

    Starting an in situ with the newly fixed material of Membranipora. I took 200 µL from X, X, X, X days and mixed. Since samples were fixed with 3.7% PFA + 0.2% Gluta I am testing the time in proteinase K for 2 genes, Synaptotagmin and Nanos (a strong one and a known one), with 30s and 1:30s.

    30s 1m30s
    Mm gene Syt1a Syt1a
    Mm gene Nanos Nanos


    Started developing at 11h30. By night a weak background began to appear in the shell.


    Change AP at 10h. Background became stronger and apparently no real signal neither in syt or nos. Some larvae in the syt wells do not have much background. Crystals appeared in the afternoon… Kept developing at 4 °C.


    Stopped developing.


    Checked under microscope… only background it seems.

  • Bruno Vellutini 18:28 on 2012/08/27 Permalink
    Tags: , , , nk6, , Prostheceraeus vittatus, , synaptotagmin   

    Probe synthesis for Terebratalia, Membranipora and Prostheceraeus 

    Started a probe pcr with the following minipreps:

    Tt gli BV157 T7 3312,97
    Tt smo BV153 SP6 1154,37
    Tt nk6 BV173 SP6 1179,23
    Mm syt1a BV86 SP6 719,42
    Mm syt1b BV89 T7 942,87
    Pv syt1b BV189 T7 921,73
    Pv foxg BV191 SP6 1464,56
    Pv syt4a BV194 T7 1354,31

  • Bruno Vellutini 23:50 on 2012/04/01 Permalink
    Tags: , , , , , , synaptotagmin   

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

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


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


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


    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, , , synaptotagmin   

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

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