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  • Bruno Vellutini 10:54 on 2014/11/27 Permalink
    Tags: , , gli, , , , , , 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 19:31 on 2014/11/18 Permalink
    Tags: gli, , , , , , , ,   

    Probe synthesis of Novocrania Hedgehog pathway 


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    Selected minipreps for probe synthesis of Novocrania Hedgehog pathway and remaining Terebratalia genes. From this post.

    BV326 Terebratalia transversa Lmx1 T7 ok + SP6
    BV328 Terebratalia transversa NK1a T7 ok + SP6
    BV330 Terebratalia transversa NK1b T7 ok + SP6
    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    TTR Terebratalia Pax6 T7 DIG
    TTR Terebratalia Pax6 T7 DNP

    Set probe PCR to run overnight.

    19/11/14

    Ran gel and painfully extracted the PCR…

    2014-11-19 19.09.46

    20/11/14

    Started probe synthesis for the genes above at 9:30. Finished at 16:30 and precipitated overnight.

    21/11/14

    gene ng/µL goal concentration volume total volume volume of hybe buffer
    Ttra lmx1 906 50 24 426 402
    Ttra nk1a 974 50 24 458 434
    Ttra nk1b 1098 50 24 516 492
    Nano ptc1 1075 50 24 505 482
    Nano smo1 2041 50 24 959 936
    Nano smo2 1830 50 24 860 836
    Nano gli 488 50 24 229 206
    Nano pax6 1350 50 24 634 611
    Nano pax37 2224 50 24 1045 1022
    Nano nk1 1050 50 24 494 470
    Ttra pax6 dig 1076 50 24 506 482
    Ttra pax6 dnp 50 50 24 23 0

     

     
  • Bruno Vellutini 18:49 on 2014/11/11 Permalink
    Tags: gli, , , ,   

    Transformation to sequencing of Novocrania 


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    I selected the 8 most important Novocrania genes from the first and second batched of cloning: ptc1, ptc2, smo1, smo2, gli, pax6, pax3/7, nk1

    Transformed and plated.

    12/11/14

    Set colony PCR:

    2014-11-12 15.43.52

    13/11/14

    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV333 Novocrania anomala Patched1 T7 ok + SP6
    BV334 Novocrania anomala Patched2 T7 ?
    BV335 Novocrania anomala Patched2 T7 ?
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV337 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV339 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV341 Novocrania anomala Gli T7 ok T7
    BV342 Novocrania anomala Pax6 T7 ok T7
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV344 Novocrania anomala Pax3/7 T7 ok T7
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    BV347 Novocrania anomala NK1 T7 ok + SP6

     

     
  • Bruno Vellutini 16:15 on 2014/11/10 Permalink
    Tags: , gli, ,   

    Cloning Hedgehog components of Novocrania 


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

    2014-11-10 22.32.27

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

     
  • Bruno Vellutini 13:33 on 2013/06/01 Permalink
    Tags: , , gli, ,   

    Beginning a double in situ with en dnp… 


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    Beginning a double in situ with:

    en (dnp) + gli (dig)
    en (dnp) + fgf8/17/18 (dig)

    02/06/13

    Added probes.

    04/06/13

    First washing day normal. Washes followed at times, but second 0.2x SSC took 40 instead of 20 min. Added Anti-Dig-POD at 1:250 concentration.

    05/06/13

    Second washing day normal.

    06/06/13

    Developed first probe. CAOS after detergent wash, during subsequent PTw washes the tape glued to the cover and FLIPPED the 4-well dish… I recovered most embryos and many remained in their wells, but for sure there has been some mixing. I added the recovered embryos to a third well.

    Apart from that the first probe works. And incubated with Anti-DNP at 1:100.

    07/06/13

    Regular washes.

    08/06/13

    Developed second probe and let it in PTw in the cold room until 12/06/13.

    12/06/13

    DAPI staining for 20 min, one wash of PTw and glycerol overnight.

     
  • Bruno Vellutini 15:54 on 2012/09/14 Permalink
    Tags: gli, , , , ,   

    In situ of Terebratalia wnt genes 


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    Starting in situ for wnt genes and segmentation genes:

    wnt1 wnt9 gli
    wnt5 wnt10 smo
    wnt6 wnt16 nk6

    All genes took 2 days to developed, except for wnt9, wnt10 and smo. I let them all in AP -MgCl2 for the weekend and resumed developing on Monday morning. I exchanged the 3 wells for new AP substrate and let overnight at 4 °C.

     
  • Bruno Vellutini 18:28 on 2012/08/27 Permalink
    Tags: , gli, , nk6, , Prostheceraeus vittatus, ,   

    Probe synthesis for Terebratalia, Membranipora and Prostheceraeus 


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    Started a probe pcr with the following minipreps:

    Tt gli BV157 T7 3312,97
    Tt smo BV153 SP6 1154,37
    Tt nk6 BV173 SP6 1179,23
    Mm syt1a BV86 SP6 719,42
    Mm syt1b BV89 T7 942,87
    Pv syt1b BV189 T7 921,73
    Pv foxg BV191 SP6 1464,56
    Pv syt4a BV194 T7 1354,31

     
  • Bruno Vellutini 12:52 on 2012/05/10 Permalink
    Tags: , , gli, , , , , , ,   

    Cloning Mm six3/6 and Tt gli, en, dlx, nk6, smo, piwia, wnt7 


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    Started a regular PCR for (re-doing this genes from scratch):

    Mm: six3/6
    Tt: piwia, engrailed, distal-less, gli, nk6, smoothened, wnt7

    Tt bands were not good, so trying a re-PCR overnight.

    11/05/12

    In the end the re-PCR did not worked well, so I purified the DNA directly for  Mm six3/6 and Tt piwia, nk6, and smoothened. Ligation was set for these and let over the weekend.

    14/05/12

    Heatshock transformation and plating at 18h.

    15/05/12

    Colony PCR and selected colonies to grow at 17h30. Tt gli is being grown from Katrine’s plate. Bands were kind of variable… do not like this; let’s see if it is contamination.

    BV143 Membranipora membranacea Six3/6 T7
    BV144 Membranipora membranacea Six3/6 T7
    BV145 Membranipora membranacea Six3/6 T7
    BV146 Terebratalia transversa Piwia T7
    BV147 Terebratalia transversa Piwia T7
    BV148 Terebratalia transversa Piwia T7
    BV149 Terebratalia transversa Piwia T7
    BV150 Terebratalia transversa NK6 T7
    BV151 Terebratalia transversa NK6 T7
    BV152 Terebratalia transversa NK6 T7
    BV153 Terebratalia transversa Smoothened T7
    BV154 Terebratalia transversa Smoothened T7
    BV155 Terebratalia transversa Smoothened T7
    BV156 Terebratalia transversa Gli T7
    BV157 Terebratalia transversa Gli T7
    BV158 Terebratalia transversa Gli T7

    18/05/12

    Set a sequencing PCR at 11:40; delivered at 19:45.

    23/05/12

    Many Wnts mixed with the proper genes again… Interestingly, Gli samples, which I prepared the minipreps directly from Katrine’s plate did not have Wnts. This indicates that the contamination event happened afterwards, possibly during Henrike’s stay. It seems that something in the ligation kit is the culprit.

     
  • Bruno Vellutini 17:20 on 2012/04/27 Permalink
    Tags: gli, , , , , , , ,   


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    Sequencing for Terebratalia segmentation genes cloned by Katrine/Andi. Just ran a sequencing PCR and sent the products to the sequencing facility.

    03/05/12

    Quickly checked the sequences today and they look bad. The only genes properly cloned were Patched and Netrin. The single Netrin sequence looks ok while the 2 Patched ones are a bit trashed.

     
  • Bruno Vellutini 18:43 on 2012/04/13 Permalink
    Tags: gli, , , , , , , , ,   

    Katrine/Andi Terebratalia segmentation genes, miniprep specs 


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    Since the previous sequencing for these minipreps did not work I am trying to sequence them again. These are the reads from the minipreps, they look ok.

    Gene ID ng/µL
    Tt gli 1 BV105 569.21
    Tt gli 2 BV106 755.79
    Tt gli 3 BV107 858.49
    Tt net 1 BV108 1080.2
    Tt net 2 BV109 416.45
    Tt net 3 BV110 452.18
    Tt net 3 BV111 474.99
    Tt nk6 1 BV112 1898.64
    Tt nk6 2 BV113 1217.41
    Tt nk6 3 BV114 1002.05
    Tt ptc 1 BV115 577.31
    Tt ptc 2 BV116 588.24
    Tt ptc 2 BV117 585.43
    Tt ptc 2 BV118 578.18
    Tt ptc 2 BV119 492.78
    Tt ptc 3 BV120 929.99
    Tt smo 1 BV121 631.31
    Tt smo 2 BV122 332.49
    Tt smo 3 BV123 1290.73
    Tt wnt7 1 BV124 968.39
    Tt wnt7 2 BV105 557.44
    Tt wnt7 3 BV106 2036.43
    Tt wnt7 4 BV107 554.82
    Tt wnt7 5 BV108 446.68

     
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