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  • Bruno Vellutini 16:28 on 2013/08/02 Permalink
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    Meara in situ for RNA binding proteins 


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    Starting in situ with mixed stages of Meara. 15 min protk.

    ago a ago b boule bruno a
    gus a mago orb2 staufen

    03/08/13

    Added probes.

    05/08/13

    First day of washes. Some small juveniles have ripped tissues. I guess the 15min protk is too much for them, the rest looks ok.

    06/08/13

    Regular 5 PBT and 5 PTw antibody washes. Kept rocking in cold room.

    07/08/13

    Developing day. Stopped argonaute after ~3h, they were becoming too dark. Bruno, Staufen are clearly there. Boule is weaker, but something. The rest there is nothing. Putting new AP in cold room.

    08/08/13

    In addition to Argonaute I also stopped Bruno, Staufen and Orb2 before going to the collection trip. The rest are developing overnight a bit more (also included Orb2 to develop overnight.

     
  • Bruno Vellutini 11:26 on 2013/05/21 Permalink
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    Probe synthesis for Meara RNA binding proteins 


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    Began the probe PCR for RNA binding proteins in Meara. Finished by May 24th.

    Gene ID ENZYME ng/µL
    Ms gus a BV226 T7 3409
    Ms boule BV229 SP6 501
    Ms staufen BV233 SP6 1141
    Ms ago a BV237 SP6 563
    Ms ago b BV238 SP6 1236
    Ms bru a BV242 SP6 1226
    Ms mago nashi BV244 SP6 336
    Ms orb2 BV247 T7 473

    Ms RNA binding

     
  • Bruno Vellutini 17:10 on 2013/03/25 Permalink
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    Cloning of Terebratalia wnt2 and other Meara RNA binding proteins 


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    Started a PCR with Terebratalia wnta, wnt2; and Meara ago a, ago b, ago c, bru a, bru b, mago, mex3, orb2.

    2013-03-26 11.47.59 2013-03-28 15.23.26

    12/04/13

    Put colonies to grow.

    Colony T7
    Ago a c1 BV235
    Ago a c2 BV236
    Ago a c3 BV237
    Ago b c1 BV238
    Ago b c2 BV239
    Ago b c4 BV240
    Bru a c1 BV241
    Bru a c2 BV242
    Bru a c3 BV243
    Mago c1 BV244
    Mago c2 BV245
    Mago c3 BV246
    Orb2 c1 BV247

     
  • Bruno Vellutini 14:36 on 2013/03/08 Permalink
    Tags: , , , , , mago nashi, , , ,   

    Cloning of Meara RNA binding proteins 


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    Initial cloning of Meara RNA binding proteins. Selection: ago a, ago b, ago c, boule, bru a, bru b, gus a, mago, mex3, orb2, stau.

    10/03/13

    Only 3 genes had visible bands: boule, gus a, and stau:

    2013-03-10 17.12.53

    Ligated these overnight.

    11/03/13

    Transformation and plated the genes.

    12/03/13

    Colony PCR with 6 colonies from each:

    2013-03-12 16.58.53

    14/03/13

    Put colonies to grow.

    boule c1 BV226 BV229
    boule c2 BV227 BV230
    boule c3 BV228 BV231
    gus a c1 BV229 BV226
    gus a c2 BV230 BV227
    gus a c3 BV231 BV228
    stau c1 BV232
    stau c2 BV233
    stau c3 BV234

    15/03/13

    Extracted plasmid and did sequencing PCR for all using T7 primer. Dropped tubes into sequencing facility.

    26/03/13

    Checking sequences and they look good, but there is a problem! Minipreps BV226, 227, 228 aligned with gus a and not with boule!!! Check why…

    Screen Shot 2013-03-26 at 10.22.12 AM

     
  • Bruno Vellutini 17:48 on 2012/05/15 Permalink
    Tags: , , , mago nashi, , , , , , ,   

    Probe synthesis for Terebratalia segmentation 


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    Beginning probe synthesis for:

    Tt netrin BV110 SP6
    Tt patched BV115 SP6
    Tt hedgehog BV129 SP6
    Tt smoothened2 BV131 SP6
    Tt nk2.2 BV135 SP6
    Tt nk5 BV139 SP6
    Mm ago2 BV25 SP6
    Mm mago nashi BV27 T7
    Mm germ cell less BV30 T7

    Run the PCR and letting overnight to extract tomorrow.

    16/05/12

    Ran gel and extracted the bands. Looking ok:

    18/05/12

    Started probe synthesis reaction at 10:40. Precipitated around 16h.

    19/05/12

    Terebratalia probes had no pellet (but in fact, I don’t know if I centrifuged it). Membranipora using SP6 had a small pellet and the two T7 ones had a huge pellet. Specs are:

    Gene ng/µL
    Mm Ago2 293.92
    Mm Magoh 3806.73
    Mm GMCL 3147.70

    Diluted to 50 ng/µL and stored at -20 °C.

    21/05/12

    Re-doing the transcription reaction for Terebratalia segmentation genes. Started at 11h, but only put at 37°C at 13h (my bad…). Precipitated at 17h30 using only 5 µL of Lithium Chloride. Could not see any pellet…

    22/05/12

    After centrifuging a thin pellet appeared in the bottom of the tubes. I used Low yield, but enough for making probes:

    Gene ng/µL
    Tt netrin 120.56
    Tt patched 174.64
    Tt hedgehog 120.93
    Tt smo2 107.95
    Tt nk2.2 211.83
    Tt nk5 221.16

    Diluted to 50 ng/µL and stored in the freezer.

     
  • Bruno Vellutini 12:45 on 2012/03/22 Permalink
    Tags: , , , mago nashi, , , ,   

    Probe synthesis for Membranipora and Priapulus 


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    T7: BV27 (MmMAGOH), BV30 (MmGMCL1), BV34 (PcTudor)
    SP6: BV25 (MmAGO2), BV28 (dicer1), BV35 (PcMaelstrom)

    DID NOT HAPPEN!

     
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