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

    Terebratalia in situ for FGFR, Frizzleds and others 

    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: aldh2, , , , , , ,   

    Probe synthesis Terebratalia 

    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.

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

    Brachiopod cloning 

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

    01/08/13

    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.

    02/08/13

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

    2013-08-02 18.29.52

    03/08/13

    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.

    05/08/13

    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

    06/08/13

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

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

    07/08/13

    Colony gel, putting good colonies to grow today.

    2013-08-07 12.43.32

    08/08/13

    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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