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  • Bruno Vellutini 19:19 on 2014/11/06 Permalink
    Tags: , , , wnt   

    Testing new Novocrania cDNA 

    I’m testing the new cDNA for Novocrania made by Daniel. He made a mix of extractions from different stages and diluted 1:10 and 1:20. I tested with previous genes that worked, pax2/5/8 and wnt11. Set the PCR with a reaction for the 1x, 1:10 and 1:20.

    07/11/2014

    2014-11-07 12.17.42

     
  • Bruno Vellutini 15:38 on 2014/09/20 Permalink
    Tags: , , , wnt   

    Membranipora Endo-IWR-1 

    Trying the Endo-IWR-1 to inhibith the Wnt Pathway. Setting 2 plates with 10, 20, and 40 µM.

    For 24h

    DMSO 10 µM
    20 µM 20 µM

    For 48h

    DMSO 10 µM
     40 µM 40 µM

    I miss calculated the well and put 20 µM on the last well of the first plate. So I set 40 µM for the third and fourth wells of the second plate.

    21/09/14

    I checked the embryos and it is hard to see if there is any phenotype. I’m letting it go another day to fix.

    22/09/14

    Fixed at 48h.

     
  • Bruno Vellutini 15:34 on 2014/09/20 Permalink
    Tags: , , , wnt   

    Membranipora Azakenpaullone for in situ 

    Prepared large spawning of Membranipora for Azakenpaullone treatment at 1, 2.5 and 5 µM in 4 mL volume of sea water.

    DMSO 1 µM
    2.5 µM 5 µM

    To be fixed at 24 and 48h.

     
  • Bruno Vellutini 16:34 on 2014/09/17 Permalink
    Tags: , , , , wnt   

    Membranipora Wnt inhibitors 

    Second round of testing inhibitors and activators of the Wnt pathway in Membranipora. Firt one was last year and they all showed a disrupted phenotype. I am repeating some treatments. Started with 2h post activation. 15 °C.

    Azakanpaullone (x2)

    DMSO 1 µM Azakanpaullone
    2.5 µM Azakanpaullone 5 µM Azakanpaullone

    IWR-1

    DMSO 10 µM IWR
    20 µM IWR 40 µM IWR

    PNU74654

    DMSO 1 µM PNU
    10 µM PNU 25 µM PNU

     18/09/14

    Fixed one plate of Azakanpaullone at 24h. Leaving the others to be fixed at 48h.

    Azak: seems to give a concentration dependent phenotype, but it is quite hard to identify what is wrong.

    IWR & PNU: Although higher concentrations seem to have disturbed the development somehow, it is not clear what is wrong. Maybe there is nothing wrong. I’m leaving them for 48h, maybe a phenotype becomes clearer.

    19/09/14

    Azak: 1 µM has a mild phenotype with a reduced early larva. 2.5 µM shows ciliated swimming balls or some partly differentiated reduced larvae. I am not sure if it is a ventralized phenotype, but it seems that the apical organ is missing. 5 µM balls of cells, not swimming, maybe already dead.

    IWR & PNU: Not sure if there is any phenotype…

    Fixed all for 1 hour at room temperature.

     
  • Bruno Vellutini 18:43 on 2014/07/01 Permalink
    Tags: , , , , wnt   

    Cloning genes that failed 

    Mmem wnt4a Nano hox1 Nano wnt4 Nano wnt7

    PIX

    Well, not that different… Membranipora gene worked somehow, a weak band.

    Set a ligation for Mmem wnt4a and Na hox1.

    03/07/14

    Heat shock transformation and plating.

     
  • Bruno Vellutini 10:11 on 2014/07/01 Permalink
    Tags: , , wnt   

    Probe synthesis for Membranipora wnts 

    Setting up a probe PCR for the synthesis of the remaining Membranipora wnt genes’ probes.

    gene id primer ng/µL
    Mmem wnt6 BV312 SP6  575
    Mmem wnt7 BV315 SP6  410
    Mmem wnt8 BV318 SP6  877
    Mmem wnt10a BV321 SP6  963
    Mmem wnt10b BV324 SP6  1089

    10/07/2014

    Done.

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

    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: , , , , , , wnt   

    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: , , , , , wnt   

    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 14:34 on 2014/01/16 Permalink
    Tags: , , , wnt   

    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

     
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