Tagged: Terebratalia transversa Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 19:26 on 2014/11/18 Permalink
    Tags: , , , , , , ovo, , , Terebratalia transversa   

    Transformation for remaining Terebratalia and Novocrania 

    Terebratalia: ovo, serrate, pygopus, legless (from here)

    Novocrania: notch, delta, gbx (from here)

    Transformed and plated with 400 µL.

    19/11/14

    Colony PCR.

    2014-11-19 19.09.53

    Set Nano delta and notch to grow.

    20/11/14

    Extracted minipreps for Nano notch and delta:

    gene id seq primer antisense
    Nano delta BV348 T7 SP6
    Nano delta BV349 T7 SP6
    Nano notch BV350 T7 SP6
    Nano notch BV351 T7 T7

    Set sequencing PCR for them.

    24/11/14

    Collected sequences. See orientation above.

     
  • Bruno Vellutini 18:02 on 2014/11/06 Permalink
    Tags: , , , opa, , rhomboid, , Terebratalia transversa   

    Cloning additional segment polarity in Terebratalia 

    Cloning of further Terebratalia segment polarity genes: opa, ovo, rho, serrate and genes I saw at the EuroEvoDevo: legless, pygopus.

    07/11/2014

    2014-11-07 12.17.29

     
  • Bruno Vellutini 17:31 on 2014/09/09 Permalink
    Tags: , , , , , Terebratalia transversa   

    Round of Terebratalia cloning 

    I have ordered additional primers to clone more segmentation genes. Setting a PCR with 25 µM for primer concentration and 65 °C for annealing temperature.

    Ttra lmx1, nk1a, nk1b, odd

    10/09/14

    2014-09-10 12.10.25

    So, odd failed.

    Set ligation at 4 °C to go overnight (have to write…)

     
  • Bruno Vellutini 16:03 on 2014/06/06 Permalink
    Tags: , , , , , Terebratalia transversa,   

    Brachiopod in situ 

    In situ for additional Terebratalia frizzleds and repeating some Novocrania for better pictures.

    Nano engrailed wnt1 wnt5 dlx pax258
    Ttra frizzled 5/8 frizzled 7 dcr1b tdr1 ago

    Started in situ with standard procedures. 8 min protk for Novocrania and 10 min for Terebratalia.

    07/06/14

    Added probes, embryos look good.

    09/06/14

    Washing day. Everything went as usual and put antibody around 18h.

    10/06/14

    Washed with PBT several times berween 10-15 min. Then PTw 30 min washes. Started developing.

    Stopped Nano dlx after 2h30min because it was done, as well as Ttra fz5/8. The rest continued developing. fz7 got pinkish really fast

    11/06/14

    fz7 and ago got really dark. I don’t know if it is background or signal, even though it looks like there is real signal within the background. I stopped them as well as engrailed and pax258, which look better than the last in situ.

    12/06/14

    Stopped wnt1, wnt5 dcr1b, tdr1. Novocrania samples are looking dark ugly already…

    13/06/14

    Trying to clear out some of the background from fz7 and ago… letting in ethanol for many hours.

     
  • Bruno Vellutini 17:01 on 2014/04/29 Permalink
    Tags: , , , , , Terebratalia transversa,   

    Cloning Ttra frizzled, Nano hox1, dlx, wnts 

    Need to clone the remaining frizzleds from Terebratalia and wnts from Novocrania. Set overnight regular PCR with:

    Nano hox1 Nano wnt4 Nano wnt7 Nano dlx Ttra fz5/8 Ttra fz7

    30/04/14

    20140430_175703

    Unfortunately hox1 and the wnts did not work. I should change something in the PCR. I extracted dlx and frizzleds and set a ligation for 4h at RT.

    Transformed and plated the 3 genes and incubated at 37 °C.

    01/05/14

    Pic colonies for colony PCR. Run gel and set bacteria to grow.

    20140501_150448

    02/05/14

    Extracted minipreps and set sequencing PCR.

     
  • Bruno Vellutini 08:00 on 2014/04/05 Permalink
    Tags: , , , , Terebratalia transversa,   

    Terebratalia Azakenpaullone treated in situ wnt1 and engrailed 

    Starting in situ with azakenpaullone-treated Terebratalia embryos with wnt1.

    Blastula Mid Blastula Mid blastula to larva Early Gastrula Early Larva
    1 µM 1 µM 1 µM 1 µM 1 µM
    10 µM 10 µM 10 µM 10 µM 10 µM
    C- C- C- C- C-

    Prepared fresh PTw and went up to hybe step. Then split samples for wnt1 and engrailed (30 wells in total). Wnt1 probe will be fine, but engrailed will be much less concentrated, probably around 0.1 ng/µL. Unfortunately this is what I had in stock.

    06/04/14

    I fell face to the ground and could not go to the lab. Chema put my 2 plates in the freezer.

    07/04/14

    I put the plates back at 62 °C for 5h. I diluted wnt1 probes and added the usual 500 µL volume to the wells. For the engrailed probe I had to dilute it to 0.8 ng/µL and use only 250 µL of volume… but well.

    09/04/14

    Washing day. Everything went normally. I added the antibody around 17:30.

    10/04/14

    Washed 2 x 15 min and 1x 1h with PBT and then 1x 15 min. Finally 5x 30 min with PTw. Left the plates in the cold room.

    11/04/14

    Developing day. Ran for 5h and some signal is appearing; stronger in the treated embryos than in the controls. Exchanged the AP substrate at 17:30 and put in the cold room.

    12/04/14

    Most of the treated embryos have a nice signal. Controls are surprisingly faint, though. I developed through the whole day and kept in the cold room overnight. Probably stopping tomorrow some wells.

    13/04/14

    Stopped the reactions in the treated samples with 3 washes of AP without magnesium chloride. I’m still developing the controls.

    14/04/14

    Still developing controls. Stopped wnt1, but not engrailed.

    15/04/14

    Exchanged again the AP. It looks much better now, signal is stronger. I did not filter the AP this time to see if I get crystals.

    Developed for 8h and stopped the reaction on the last wells.

    16/04/14

    Ethanol washes upt to glycerol.

     
  • Bruno Vellutini 16:46 on 2014/03/14 Permalink
    Tags: , , Terebratalia transversa   

    Defined semester plan with Andi. Work on brachiopod segmentation paper and analyse data for the bryozoan spiral cleavage.

     
  • Bruno Vellutini 23:20 on 2014/01/16 Permalink
    Tags: , Terebratalia transversa   

    Azakenpaullone for bilateral gastrula 

    Set Azakanpaullone treatment for bilateral gastrula to see how it affects lobe development.

    CTRL FSW 1 µM Az 10 µM Az

     
  • Bruno Vellutini 14:34 on 2014/01/16 Permalink
    Tags: , , Terebratalia transversa,   

    Vivo-Morpholino next with pronase and electroporation 

    Considering my last observations of the vivo-morpholinos I’m setting up a new experiment using a lower concentration of pronase, electroporating twice (because I could not figure out how to do longer pulses), and properly washing out the pronase after 1h. Default settings:

    Vivo-Morpholino: 25 µM
    Pronase: 5 µg/mL
    Washing after: 1h
    Electroporation: 50 V, 25 µF, 1000 resistance
    Actual pulses:

    CTRL FSW CTRL Pronase EN TB Pronase EN SB Pronase WNT1 TB Pronase WNT1 SB Pronase
    CTRL Electroporation EN TB Electroporation EN SB Electroporation WNT1 TB Electroporation WNT1 SB Electroporation

     
  • Bruno Vellutini 20:17 on 2014/01/15 Permalink
    Tags: , , Terebratalia transversa,   

    Terebratalia Vivo-Morpholino engrailed/wnt1 with permeabilization 

    Use 25 µM concentration for vivo-morpholinos.

    CTRL FSW
    CTRL CFSW 1h Ttra_en_TB_vMO 25 µM Ttra_en_SBe2i2_vMO 25 µM Ttra_wnt1_TB_vMO 25 µM Ttra_wnt1_SBe2i2_vMO 25 µM
    CTRL Pronase 10 µg/mL 1h Ttra_en_TB_vMO 25 µM Ttra_en_SBe2i2_vMO 25 µM Ttra_wnt1_TB_vMO 25 µM Ttra_wnt1_SBe2i2_vMO 25 µM
    CTRL Electroporation in CFSW Ttra_en_TB_vMO 25 µM Ttra_en_SBe2i2_vMO 25 µM Ttra_wnt1_TB_vMO 25 µM Ttra_wnt1_SBe2i2_vMO 25 µM

    Electroporation setup

    BioRad Gene Pulser 50V, 25 µF, resistance set to infinity.

    [photo]

    ms
    ctrl 1.1
    en tb 1.0
    en sb 1.1
    wnt1 tb 1.2
    wnt1 sb 0.9

    16/01/14

    • Calcium-free seawater look normal trilobed.
    • Pronase treatment embryos failed to gastrulate, including the control.
    • Electroporation look normal except for wnt1 sb which seem either delayed or did not manage to close the blastopore.
     
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