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  • Bruno Vellutini 19:15 on 2013/03/08 Permalink
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    Double in situ in Terebratalia 


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    Starting double in situ with probes correctly diluted to 1 ng/µL. Stages sampled are: late blastula, early/mid/late gastrula, trilobed, early/late larva.

    en-DNP+wnt1-DIG-POD en-DNP+wnt5-DIG-POD

    Regular beginning. I left 5 minutes of the ProtK shaking, mistakenly.

    11/03/13

    Regular washing. Put Anti-DIG-POD diluted 1:250 (in 250 µL each well).

    12/03/13

    Began washing at 11h30. Did extra wash with PBT and also a couple of extras for PTw.

    13/03/13

    Developed first probe with Cy3 and stopped the reaction with 1 detergent wash of 5 min and another detergent wash of 10 min. Specific expression can be seen in the stereoscope. Put the Anti-DNP antibody for the second probe.

    14/03/13

    Just washed thoroughly.

    15/03/13

    Develop the second.

    WORKS! engrailed=green, wnt1=red

    en+wnt1

     
  • Bruno Vellutini 14:36 on 2013/03/08 Permalink
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    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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