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  Bruno Vellutini 18:29 on 2015/04/29 Permalink
  Tags: bmp ( 3 ), chordin ( 3 ), cloning, 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

   

   
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  Bruno Vellutini 13:36 on 2015/01/19 Permalink
  Tags: cloning, Membranipora membranacea ( 140 ), Novocrania anomala ( 43 ), patched ( 12 ), pcr ( 37 ), smoothened ( 17 ), wnt ( 60 )   

  Cloning more segmentation genes 

  I selected alternate primers for getting longer fragments and improving the quality of Novocrania in situs.

  Nano ptc2, wnt1, smo1, smo2, fgf8, Mmem wnt4

  

  wnt1 did not work again, bad ptc2 again, smo1 and smo2 weakly worked, fgf8 worked, Mmem wnt4 not… Re-run Nano ptc2 with different primers:

  

  Combination of primers 3F and 2R (395 bp fragment) was the only one that worked. Transformed:

  

  	T7
  Na wnt5	BV352
  Na wnt5	BV353
  Na smo2	BV354
  Na fgf8	BV355
  Na fgf8	BV356
  Na ptc2	BV357
  Na ptc2	BV358

   

   
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  Bruno Vellutini 15:31 on 2014/11/25 Permalink
  Tags: cloning, Novocrania anomala ( 43 ), pcr ( 37 ), wnt ( 60 )   

  Cloning Novocrania patched and longer clones of wnt1, wnt5 

  I made new primers for ptc2 and longer clones for wnt1 and wnt5. Set PCR overnight.

  26/11/14

  Ran gel and cut a band, but wnt1 and ptc2 did not work… OF COURSE! because fragment lengths are the following: wnt1=1239 bp, wnt5=1097 bp, ptc2=1258 bp! And the elongation time of the PCR was set to X.

  

  28/11/14

  Re-tried the same PCR with a longer elongation time. The result was the same. Chema suggested running nested primers to see if something appears.

  

   
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  Bruno Vellutini 19:26 on 2014/11/18 Permalink
  Tags: cloning, delta ( 4 ), gbx ( 3 ), legless ( 2 ), notch ( 4 ), Novocrania anomala ( 43 ), ovo, pygopus ( 2 ), serrate ( 2 ), Terebratalia transversa ( 79 )   

  Transformation for remaining Terebratalia and Novocrania 

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

  Novocrania: notch, delta, gbx (from here)

  Transformed and plated with 400 µL.

  19/11/14

  Colony PCR.

  

  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.

   
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  Bruno Vellutini 16:15 on 2014/11/10 Permalink
  Tags: cloning, gli ( 10 ), patched ( 12 ), smoothened ( 17 )   

  Cloning Hedgehog components of Novocrania 

  PCR with diluted primers for 40 cycles instead of 34 for: ptc1, ptc2, smo1, smo2, gli

  

  Had two bands for some. Set a ligation overnight for all at 4 °C.

   
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  Bruno Vellutini 19:19 on 2014/11/06 Permalink
  Tags: cloning, Novocrania anomala ( 43 ), pax ( 16 ), wnt ( 60 )   

  Testing new Novocrania cDNA 

  I’m testing the new cDNA for Novocrania made by Daniel. He made a mix of extractions from different stages and diluted 1:10 and 1:20. I tested with previous genes that worked, pax2/5/8 and wnt11. Set the PCR with a reaction for the 1x, 1:10 and 1:20.

  07/11/2014

  

   
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  Bruno Vellutini 18:02 on 2014/11/06 Permalink
  Tags: cloning, legless ( 2 ), odd ( 2 ), opa, pygopus ( 2 ), rhomboid, serrate ( 2 ), Terebratalia transversa ( 79 )   

  Cloning additional segment polarity in Terebratalia 

  Cloning of further Terebratalia segment polarity genes: opa, ovo, rho, serrate and genes I saw at the EuroEvoDevo: legless, pygopus.

  07/11/2014

  

   
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  Bruno Vellutini 17:31 on 2014/09/09 Permalink
  Tags: cloning, lmx ( 3 ), nk ( 16 ), odd ( 2 ), pcr ( 37 ), Terebratalia transversa ( 79 )   

  Round of Terebratalia cloning 

  I have ordered additional primers to clone more segmentation genes. Setting a PCR with 25 µM for primer concentration and 65 °C for annealing temperature.

  Ttra lmx1, nk1a, nk1b, odd

  10/09/14

  

  So, odd failed.

  Set ligation at 4 °C to go overnight (have to write…)

   
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  Bruno Vellutini 18:43 on 2014/07/01 Permalink
  Tags: cloning, hox ( 3 ), Membranipora membranacea ( 140 ), Novocrania anomala ( 43 ), wnt ( 60 )   

  Cloning genes that failed 

  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.

   
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  Bruno Vellutini 18:36 on 2014/06/06 Permalink
  Tags: cloning, Membranipora membranacea ( 140 ), pcr ( 37 )   

  Cloning Membranipora Wnts 

  Set PCR.

  07/06/14

  Gel, extraction, ligation, transform.

  09/06/14

  Set colony PCR overnight.

  10/06/14

  Ran gel and put colonies to grow.
  

  11/06/14

  Extracted minipreps and did sequencing PCR. Left samples at the sequencing facility.