PhD notebook

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  Bruno Vellutini 10:36 on 2013/12/16 Permalink
  Tags: confocal ( 23 ), engrailed ( 39 ), in situ ( 43 ), Novocrania anomala ( 43 )   

  Propidium+Phallacidin and Murray Clear for Novocrania engrailed fluorescent in situ 

  
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  Since mounting with TDE was not so good (crystals and some weird noisy background) I am staining with propidium iodide and phallacidin and mounting with murray clear. This should give a clean embryo with the mesodermal signal visible enough for details. Fluorochrome for engrailed is Cy5.

  17/12/13

  Confocal.

   
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  Bruno Vellutini 13:17 on 2013/12/08 Permalink
  Tags: in situ ( 43 ), Novocrania anomala ( 43 ), pax ( 16 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Terebratalia and Novocrania in situ 

  
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  Began in situ 10 min protk (half-concentrated for Nano) with:

  Ttra wnt2	Ttra wnta
  Nano wnt8	Nano wnt11	Nano pax2/5/8

  07/12/13

  Added probes at 13:00.

  09/12/13

  First day of washes, added anti-dig-ap around 18h.

  10/12/13

  Washing off antibody, starting with 2x 15min PBT and after 1h 3x 15min PBT and normal PTw washes.

  Started developing at 16h. Some signal started to appear at Wnt11 ~18h30.

  11/12/13

  Arrived 9h30 and the wells were purple! Did an extra wash with AP substrate and let at RT. Exchanged AP 4 times and stopped the reaction at 19h with 3x AP without MgCl2 and 5x PTw. Kept at 4 °C overnight.

  12/12/13

  Ethanol washes, then first wash with glycerol and let in nutator for 3h. Exchange glycerol for glycerol+dapi and let it overnight.

  13/12/13

  Mounted slides of Novocrania and took pictures.

   
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  Bruno Vellutini 11:02 on 2013/12/04 Permalink
  Tags: Novocrania anomala ( 43 ), pax ( 16 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Probe synthesis for Novocrania and Terebratalia 

  
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  Probe PCR with 6 samples:

  

  05/12/13

  Set transcription reaction for all except for MP68 at 10h30. Began precipitation with 5 µL of lithium.

  06/12/13

  	ng/µL
  Ttra wnt2	1008
  Ttra wnta	801
  Nano wnt8	2000
  Nano wnt11	1353
  Nano pax258	860