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  • Bruno Vellutini 13:17 on 2013/12/08 Permalink
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    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.

     
  • Bruno Vellutini 11:02 on 2013/12/04 Permalink
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    Probe synthesis for Novocrania and Terebratalia 


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    Probe PCR with 6 samples:

    2013-12-04 18.40.07

    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

     
  • Bruno Vellutini 21:32 on 2013/11/26 Permalink
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    Cloning Novocrania and Terebratalia again 


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    Having the gene expression patterns of additional genes in Novocrania would be great to compare the body patterning with Terebratalia. I have selected some genes specific to apical, mantle, or pedicle lobe to try to map the expression of Terebratalia in Novocrania.

    • Apical lobe: wnt7, wnt8.
    • Mantle lobe: dlx, pax258, hox1.
    • Pedicle lobe: wnt4, wnt11.

    Also a couple of wnt genes that hadn’t worked in Terebratalia are also being cloned. I forgot to include Nano dlx in the order :/

    2013-11-26 21.29.18

    27/11/13

    Transformed and plated at 15h30. Put plates in incubator at 19h00.

    28/11/13

    Colony PCR and put cultures to grow. Wnt7 and Hox1 were not good so I plated them again with remaining bacteria.

    2013-11-28 18.31.05

    29/11/13

    Miniprep and sequencing PCR. Dropped sequences in facility.

    03/12/13

    BV275 Terebratalia transversa Wnt2 T7 ok + SP6
    BV276 Terebratalia transversa Wnt2 T7 ok + SP6
    BV277 Terebratalia transversa Wnt2 T7 ok + SP6
    BV278 Terebratalia transversa WntA T7 ok + SP6
    BV279 Terebratalia transversa WntA T7 ok + SP6
    BV280 Terebratalia transversa WntA T7 ok + SP6
    BV281 Novocrania anomala Wnt7 T7 ok
    BV282 Novocrania anomala Wnt11 T7 ok + SP6
    BV283 Novocrania anomala Wnt11 T7 ok T7
    BV284 Novocrania anomala Pax2/5/8 T7 ok + SP6

     
  • Bruno Vellutini 13:20 on 2013/10/21 Permalink
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    Terebratalia additional double in situs 


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    Started Terebratalia double in situs with the following selected probes. Everything looks ok.

    wnt1 (DNP) + wnt4 (DIG) en (DNP) + wnt7 (DIG)
    wnt1 (DNP) + wnt8 (DIG) en (DNP) + pax258 (DIG)

    22/10/13

    • Added probes to Terebratalia doubles.

    24/10/13

    • Terebratalia doubles washing day.
    • Add Terebratalia first antibody anti-dig-pod 1:250.

    25/10/13

    • Terebratalia doubles wash off first antibody.

    27/10/13

    • Terebratalia doubles develop first probe and inactivate.
    • Terebratalia doubles incubate with second antibody.

    28/10/13

    • Terebratalia doubles wash off second antibody.

    29/10/13

    • Terebratalia doubles develop second probe.
    • Terebratalia doubles stain with phallacidin.
    • Terebratalia doubles embed in TDE+DAPI.

    30/10/13

    • Mount Terebratalia doubles.

    31/10/13

    • Confocal slot for Terebratalia doubles.

    01/11/13

    Here are some scans.

     
  • Bruno Vellutini 17:33 on 2013/09/13 Permalink
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    Terebratalia in situ for FGFR, Frizzleds and others 


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    Starting an in situ for additional Terebratalia genes related to the segmentation project. Also a double with wnt1+wnt5.

    FGFR Pax2/5/8 Frizzled2 Frizzled4
    Frizzled10 LFNG ALDH2 Wnt1+Wnt5

    14/09/13

    Put probe.

    16/09/13

    Washed in situ and added anti-dig-ap 1:5000 to the first seven genes and anti-dig-pod 1:250 for the double fluorescent.

    17/09/13

    • Washed antibody.
    • Set Cy3 TSA to double fluorescent for 1h45.
    • Started developing regular NBT/BCIP at 16h.
    • Stopped Fz2 around 20h, let the rest overnight.

    18/09/13

    • Stopped FGFR and Fz4. Let the rest continue, almost stopped Pax2/5/8, since it is almost there.

    22/09/13

    • Ethanol washes.
    • Glycerol added.
     
  • Bruno Vellutini 16:01 on 2013/09/10 Permalink
    Tags: , , , , pax, , ,   

    Probe synthesis Terebratalia 


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    Probe PCR for Terebratalia genes (except wnt1, I had it already).

    gene id enzyme utp ng/µL
    FGFR BV249 SP6 DIG 1618.42
    Pax 2/5/8 BV251 T7 DIG 1155.38
    Frizzled 2 BV254 SP6 DIG 1542.03
    Frizzled 4 BV257 SP6 DIG 1629.45
    Frizzled 10 BV259 SP6 DIG 978.54
    LFNG BV263 SP6 DIG 1137.59
    ALDH2 BV265 T7 DIG 1213.53
    WNT1 AH23 SP6 DNP 133.85

    11/09/13

    Everything looks alright!

    2013-09-11 16.37.13

    12/09/13

    Set transcription reaction at 10:30, stopped at 18:30.

    13/09/13

    Finished probe synthesis and everything went well, very good pellets. See concentration values at the table above.

     
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