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  • Bruno Vellutini 10:54 on 2014/11/27 Permalink
    Tags: , , , , notch, , , , pou, ,   

    Novocrania and double in situs of Terebratalia 


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    Nano ptc1 Nano smo1 Nano smo2 Nano gli
    Nano pax6 Nano delta Nano notch
    Ttra pax6 (DNP/cy5) + pax258 (DIG) Ttra en (DNP) + pax6 (DIG) Ttra wnt1 (DNP) + delta (DIG) Ttra wnt1 (DNP) + pou4 (DIG)

     28/11/14

    Added probes at 1 ng/µL concentration.

    30/11/14

    Washes, incubated with the blocking for 1h and at 4 °C overnight with the respective antibodies. Anti-DIG-AP 1:5000 for all Novocrania wells and Anti-DIG-POD 1:250 for all Terebratalia wells.

    01/12/14

    Washed antibody from all samples with:

    • 5x 15 min PBT and 5x 30 min PTw.
    • 3x 5min washes of AP minus MgCl2 for regular in situ and TNT buffer for fluorescent in situs.

    Developed fluorescent wells with TSA kit using cy3 fluorochrome for 2 hours. Sropped the reaction with 5x 5 min detergent solution at 60°C followed by PTw washes. POD inactivation with 0.1% H2O2 for 45 min, PTw washes, and finally formamide based POD inactivation buffer for 15 min at 60 °C. Signal for pax6 and delta were very strong, pou4 was strong and pax258 was medium.

    I started developing Novocrania with NBT/BCIP. Pax6 was the first to come, strongly. The others were slower, but gli had a good signal/noise ratio. Others acquired background after the first AP change after 2 hours. I stopped all, except delta and smo2 (which had less background).

    02/12/14

    Today they were quite dark :/

    Went through ethanol washes and antibody washes for fluorescent. Added 70% glycerol in PTw with 1:10000 sytox green.

     
  • Bruno Vellutini 15:29 on 2014/11/25 Permalink
    Tags: , notch, ,   

    Probe synthesis Novocrania delta/notch 


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    Probe PCR set for Novocrania delta (BV348: Sp6) and notch (BV 350: Sp6).

    2014-11-25 16.44.03

    Cut and extracted.

    26/11/14

    Set transcription reaction for 7h. Added DNAse and 5 µL of lithium.

    27/11/14

    Finishing up.

    ng /µL
    Nano delta 1978
    Nano notch 1571
    Tt pou4 371

     

     
  • Bruno Vellutini 19:26 on 2014/11/18 Permalink
    Tags: , , , , notch, , ovo, , ,   

    Transformation for remaining Terebratalia and Novocrania 


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    Terebratalia: ovo, serrate, pygopus, legless (from here)

    Novocrania: notch, delta, gbx (from here)

    Transformed and plated with 400 µL.

    19/11/14

    Colony PCR.

    2014-11-19 19.09.53

    Set Nano delta and notch to grow.

    20/11/14

    Extracted minipreps for Nano notch and delta:

    gene id seq primer antisense
    Nano delta BV348 T7 SP6
    Nano delta BV349 T7 SP6
    Nano notch BV350 T7 SP6
    Nano notch BV351 T7 T7

    Set sequencing PCR for them.

    24/11/14

    Collected sequences. See orientation above.

     
  • Bruno Vellutini 18:33 on 2014/11/07 Permalink
    Tags: , , , , notch, , ,   

    Novocrania cloning 


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    Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.

    08/11/14

    Ran gel and set ligation.

    2014-11-08 15.29.44

     
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