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  • Bruno Vellutini 21:02 on 2014/07/15 Permalink
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    Anti-MAPK immuno staining Membranipora 


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    I realized that I don’t have a proper description of all Membranipora stages with the anti-mapk antibody. I prepared 4 wells with mixed stages as following:

    S1: 3h, 10h, 11h, 13h, 16h S2: 18h, 22h, 26h, 30h, 42h
    S3: 46h, 50h, 68h, 70h, 88h S4: 3d, 4d, 5d, 9d
    1. Mix samples in PTx.
    2. Exchange 4x in 30 min.
    3. 2x 15 min PBT.
    4. 1x 40 min PTx+NGS.
    5. Incubate with Anti-MAPK 1:200 in 200 µL of PTx+NGS.

    16/07/14

    1. 3x 5 min PBT.
    2. 4x 30 min PBT.
    3. 30 min PTx+NGS.
    4. 1:250 Anti-Mouse POD (Jackson) + Anti-Mouse HRP in PTx+NGS and incubate overnight in cold room at 21h.

    17/07/14

    1. Washed out secondary with 3x 5min and 4x 30 min PBT.
    2. Washed 3x 10min in TNT buffer.
    3. Develop with TSA+Cy5 for 5 min.
    4. Stopped the reaction in PTx.
    5. 45 min with 1:4000 Sytox Green.
    6. Embedding with TDE.

    I realized that sytox staining was fading very quickly under fluorescent scope and decided to add 1:2000 sytox green into TDE and leave it overnight.

    18/07/14

    Mounted and scanned sample 1 only in the confocal. They look nice, but maybe next time I would either stop the reaction with detergent solution or develop for shorter time, 3 minutes maybe.

    MAX_Mmem_MAPK_1.lif - Series003 MAX_Mmem_MAPK_1.lif - Series016 MAX_Mmem_MAPK_1.lif - Series022

     
  • Bruno Vellutini 18:43 on 2014/07/01 Permalink
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    Cloning genes that failed 


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    Mmem wnt4a Nano hox1 Nano wnt4 Nano wnt7

    PIX

    Well, not that different… Membranipora gene worked somehow, a weak band.

    Set a ligation for Mmem wnt4a and Na hox1.

    03/07/14

    Heat shock transformation and plating.

     
  • Bruno Vellutini 12:51 on 2014/07/01 Permalink
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    Membranipora 4D recording 


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    Likely normal development  at 15 °C.

    Folder: BCV_Mm_2014_07_01 (old Mmem_01_07_2014)

     
  • Bruno Vellutini 10:11 on 2014/07/01 Permalink
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    Probe synthesis for Membranipora wnts 


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    Setting up a probe PCR for the synthesis of the remaining Membranipora wnt genes’ probes.

    gene id primer ng/µL
    Mmem wnt6 BV312 SP6  575
    Mmem wnt7 BV315 SP6  410
    Mmem wnt8 BV318 SP6  877
    Mmem wnt10a BV321 SP6  963
    Mmem wnt10b BV324 SP6  1089

    10/07/2014

    Done.

     
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