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

    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 17:45 on 2013/11/23 Permalink
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    Novocrania engrailed fluorescent in situ 

    Began a fluorescent in situ of Novocrania engrailed to better understand the mesodermic expression. The only difference this time was 10 min of half-concentrated proteinase-k (1.25 µL in 5 mL) instead of 5 min.

    24/11/13

    Diluted new probe and added to the sample.

    26/11/13

    Wash day went on smoothly. Added 1:100 anti-dig-pod antibody and incubated in the cold room.

    27/11/13

    Wash off antibody.

    • Developed with TSA Cy5 for 2h.
    • Stopped reaction with 2x 5 min in detergent solution at 62 °C.
    • 1x 5 min in detergent solution at RT.
    • 3x 5 min PTw at RT.
    • Selected 6 embryos of different stages and went through TDE steps until final 97% + DAPI.
    • Letting overnight in cold room.

    28/11/13

    Crystals have formed and are sticking around the embryos… Mounted a slide and it works, although there is background.

    29/11/13

    Confocal at 8h!

    Nano en TDE 1.lif - Series004 s1 Nano en TDE 1.lif - Series004 s2 Nano en TDE 1.lif - Series004 s3

     
  • Bruno Vellutini 18:19 on 2013/11/20 Permalink
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    Membranipora MAPK U0126 quantification 

    Before staining for the phenotype description I counted the number of normal embryos in the fine picked samples. Normal means late gastrula with apical lobe and elongated body axis.

    Seawater DMSO 1µM 10µM 25µM
    Normal late gastrula 31 31 33 0 0
    Weird 6 11 10 40 23
    Total 37 42 43 40 23

     
  • Bruno Vellutini 10:16 on 2013/11/18 Permalink
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    Phallacidin staining for Novocrania and Membranipora 

    Stain Novocrania and Membranipora with phallacidin then mount in TDE and glycerol, respectively (phallacidin has to be working in Membranipora to help with the phenotype description).

    Novocrania to be mounted in TDE:

    Novocrania mixed stages

    Membranipora to be mounted in glycerol:

    Mmem control Mmem U0126 1µM
    Mmem U0126 10µM Mmem U0126 25µM

    Put in TDE and glycerol today.

    19/11/13

    Mount slides for confocal:

    • Novocrania mixed stages in TDE+DAPI.
    • Membranipora each treatment in glycerol+DAPI.
    • Novocrania failed in situs in glycerol+DAPI for morphology.

    20/11/13

    Confocal day.

     
  • Bruno Vellutini 16:35 on 2013/11/05 Permalink
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    Novocrania in situ wnts 

    Another in situ now using the newly synthesized probes.

    06/11/13

    Added Probes.

    08/11/13

    First washing day. Diluted 0.2 µL of Anti-DIG-AP in 1x blocking buffer and let overnight in cold room.

    09/11/13

    Washing off antibody. And started developing. Exchanged the substrate 4 times and no signal. Let overnight in cold room.

    10/11/13

    Wnt1 signal is visible! Ring around blastopore in gastrula and posterior ventral in later stages. No bands! Continued developing until 14h, then 4°C until 18h when I exchanged the substrate and let overnight again. Wnt5 had signal in the chaetae sacs.

    11/11/13

    Checked and signal looks a bit stronger so I developed 40 min more and stopped the reaction. Crystals were forming in the wnt5 well. Ethanol washes and will put them in glycerol with DAPI.

    12/11/13

    Mount and take pictures.

    Wnt1

    Wnt5

     
  • Bruno Vellutini 11:10 on 2013/11/04 Permalink
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    Re-synthesis of Novocrania wnt1 and wnt5 probes 

    Since the probes do not seem to be working for Novocrania in situ I will restart probe synthesis from scratch.

    • Set probe PCR.
    • Run Gel.
    • Extract.

    2013-11-04 17.04.12

    05/11/13

    Transcription reaction for 6h and precipitate at -20 °C overnight.

    06/11/13

    Finished probe synthesis:

    gene ng/µL
    wnt1 3349
    wnt5 185

     
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