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  • Bruno Vellutini 12:49 on 2015/01/30 Permalink
    Tags: , fgf8, , , , , , ,   

    In situ Novocrania and Terebratalia 

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    In situ using Nano longer probes to try to get a better signal to ratio. Also fgf8 probe, important for the brain boundary. I also need better hedgehog pictures, so I’m repeating the in situ with a new probe in Ttra and also trying to get a good early gastrula, bilateral gastrula e late larvae of engrailed.

    Nano wnt5 Nano smo2 Nano fgf8 Nano ptc1
    Ttra hh Ttra en late blastula / early gastrula Ttra en bilateral gastrula / late larva

    10 min ProtK for all. Incubated at 65 °C at 2000.


    Diluted probes to 1 ng/µL and added at 0800.


    First day of washes. Added anti-dig-ap at 1600.


    Washed off antibody with 3x 5min followed by 4x 15 min and 5x 30 min. Developed with nbt/bcip at 1600. All wells showed good signal/noise ratio. The worst is wnt5, but it is still better than the other in situ. I exchanged the AP for all wells at 1930 and stopped all except hedgehog and en bilateral (exchanged AP).


    Exchanged the AP for the two Terebratalia wells, I’ll let them develop longer.

    Stopped at 1500 and did ethanol washes. Let overnight in 70% glycerol.


    Mounting and pictures.

  • Bruno Vellutini 18:15 on 2015/01/27 Permalink
    Tags: , fgf8, , , , , ,   

    Probe synthesis of Terebratalia and Novocrania 

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    Probe synthesis of some longer fragments for Novocrania in situ wnt5, smo, fgf8 and standard Terebratalia clones hh and en.

    SP6 ng/µL
    Na wnt5 BV352  1146.5
    Na smo2 BV354  906.7
    Na fgf8 BV355  964.2
    Tt hh BV129  1510.6
    Tt en TTR54  573.1

    Set probe PCR. Extracted.


    Ran gel and extracted.

    2015-01-30 11.05.52


    Set probe synthesis and precipitated.


    Finished probes.

  • Bruno Vellutini 12:53 on 2013/07/31 Permalink
    Tags: , armadillo, , fgf8, , , , , ,   

    Brachiopod cloning 

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    Began PCR for cloning Novocrania en, wnt1, wnt5 and Terebratalia fgfr, lfng, pax2/5/8, Fz2, Fz4, Fz5/8, Fz7, Fz10, aldh2, arm.


    Gel, Novocrania genes did not work. Others did, extraction and ligation.

    2013-08-01 15.09.57

    I started a PCR for Novocrania genes using a lower annealing temperature of 55 °C.


    Managed one band for engrailed, but not at the right length, slightly shorter.

    2013-08-02 18.29.52


    I reset the ligation for: lfng, fz58 7, aldh2, arm.

    Colony PCR running for the rest.

    Also started a PCR with Nano wnt1 and wnt5 with annealing temperature of 50 °C.


    Transformed and plated the genes that did not work previously (lfng, fz5/8, fz7, aldh2, arm).

    Novocrania gel is not promising, bands are not the right size, need to design new primers.

    2013-08-05 13.24.23

    Colony PCR went well and today I’ll put the colonies to grow.

    2013-08-05 13.24.13

    Animal Gene Colony T7
    Ttra FGFR 1 BV248
    Ttra FGFR 2 BV249
    Ttra FGFR 3 BV250
    Ttra Pax 2/5/8 1 BV251
    Ttra Pax 2/5/8 2 BV252
    Ttra Pax 2/5/8 3 BV253
    Ttra Fz2 1 BV254
    Ttra Fz2 2 BV255
    Ttra Fz2 3 BV256
    Ttra Fz4 2 BV257
    Ttra Fz4 3 BV258
    Ttra Fz10 2 BV259
    Ttra Fz10 3 BV260
    Ttra Fz10 4 BV261
    Nano En 3 BV262


    Sequences above dropped at the sequencing facility after miniprep extraction and sequencing pcr.

    Also, set colony pcr for the genes that hadn’t worked.


    Colony gel, putting good colonies to grow today.

    2013-08-07 12.43.32


    Extracted minipreps for Lfng 3 and 5 and Aldh2 1. Sequencing PCR and dropped samples in the facility.

    Gene ID
    LFNG c3 BV263
    LFNG c5 BV264
    ALDH2 c1 BV265

  • Bruno Vellutini 13:33 on 2013/06/01 Permalink
    Tags: , fgf8, , ,   

    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)


    Added probes.


    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.


    Second washing day normal.


    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.


    Regular washes.


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


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

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