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  • Bruno Vellutini 17:33 on 2013/09/13 Permalink
    Tags: aldh2, , , , , , , , ,   

    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


    Put probe.


    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.


    • 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.


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


    • Ethanol washes.
    • Glycerol added.
  • Bruno Vellutini 16:01 on 2013/09/10 Permalink
    Tags: aldh2, , , , , , ,   

    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


    Everything looks alright!

    2013-09-11 16.37.13


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


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

  • Bruno Vellutini 12:53 on 2013/07/31 Permalink
    Tags: aldh2, armadillo, , , , , , , ,   

    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

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