PhD notebook

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  Bruno Vellutini 18:24 on 2015/05/08 Permalink
  Tags: bmp, chordin ( 3 ), distal-less ( 11 ), fgf ( 2 ), in situ ( 43 ), Membranipora membranacea ( 140 ), nanos ( 25 ), Novocrania anomala ( 43 ), piwi ( 32 ), pl10 ( 6 ), vasa ( 22 ), wnt1 ( 3 )   

  Membranipora in situ and Novocrania wnt1 

  Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.

  wnt1	dlx	bmp2/4	chordin	fgf
  nanos	vasa	piwi1	piwi2	pl10
  Na wnt1

  Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.

  09/05/15

  Added probes just after synthesis.

  11/05/15

  Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.

  12/05/15

  Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.

  13/05/15

  Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.

   
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  Bruno Vellutini 20:09 on 2015/05/06 Permalink
  Tags: bmp, chordin ( 3 ), fgf ( 2 ), pcr ( 37 ), piwi ( 32 ), probe ( 24 )   

  Probe PCR Membranipora final genes 

  Probe PCR for bmp/24, chordin, fgf8 and piwi1.

  07/05/15

  Extracted probe PCR gel.

  

  08/05/15

  Bit weird the bmp2/4, but well. Made the transcription reaction for 6:30h and put probes to precipitate.

  09/05/15

  Finish probes.

   
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  Bruno Vellutini 18:29 on 2015/04/29 Permalink
  Tags: bmp, chordin ( 3 ), cloning ( 15 ), fgf8/17/18 ( 4 ), Membranipora membranacea ( 140 ), nanos ( 25 ), piwi ( 32 ), vasa ( 22 )   

  Membranipora cloning final genes 

  Set PCR with:

  Mm bmp2/4	Mm otx1	Mm chordin	Mm fgf8/17/18
  Mm nanos	Mm piwi1	Mm piwi2	Mm vasa

  30/04/15

  Ran gel:

  

  Most important genes worked: bmp2/4, chordin and fgf8. piwi1 is bonus. Extract these and set a ligation for 3h. Transformed and plated around 18h.

  01/05/15

  Set colony PCR.

  

  Put colonies to grow.

  02/05/15

  Extracted minipreps, set sequencing PCR and dropped at seq facility.

  BV378	Membranipora membranacea	Bmp2/4	T7	ok	+	SP6
  BV379	Membranipora membranacea	Bmp2/4	T7	ok	+	SP6
  BV380	Membranipora membranacea	Chordin	T7	ok	+	SP6
  BV381	Membranipora membranacea	Chordin	T7	ok	+	SP6
  BV382	Membranipora membranacea	Fgf8/17/18	T7	ok	+	SP6
  BV383	Membranipora membranacea	Fgf8/17/18	T7	ok	+	SP6
  BV384	Membranipora membranacea	Piwi1	T7	ok	+	SP6
  BV385	Membranipora membranacea	Piwi1	T7	ok	+	SP6