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  • Bruno Vellutini 16:28 on 2013/08/02 Permalink
    Tags: , , , , , , , orb,   

    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
    Tags: , , , , , , orb, ,   

    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/┬ÁL
    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
    Tags: , , , , , orb, ,   

    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: , , , , , , , , orb,   

    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

     
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