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  • Bruno Vellutini 11:13 on 2015/05/12 Permalink
    Tags: , , runx, ,   

    Novocrania wnt1 in situ with longer probe 

    Na wnt1 Tt runx

    Did everything as usual and embryos looked normal.


    Embryos quite damaged, including Terebratalia. Possible that pipette is not well calibrated?

    Restarted in situ today with extreme gentleness. Added 5 µL protk in 10 mL (instead of 2.5 in 5 µL) using the P20 pipette. Fosfatase wash with 83 °C and added hybe buffer around 1800.


    Add probe.

  • Bruno Vellutini 18:48 on 2014/11/22 Permalink
    Tags: , , , , , , , runx, ,   

    Terebratalia segmentation in situ 

    In order to have more temporal resolution of engrailed I am adding one well per stage. In addition some segmentation related genes.

    en cleavage+blastula en radial gastrula en asym gastrula en bilateral gastrula en trilobed+early+late larva
    pax6 nk1a nk1b lmx1
     lfng runx hh


    Washed probe out. Added antiDIG-AP antibody at 1700 and incubated overnight at 4 °C.


    Washed antibody and started developing. Pax6 came up quite fast. I exchanged the solution after 1h and stopped after 2h. The remaining are slowly coming up, except asym and bilateral of engrailed. I used one tube of 0.8 ng/µL of probe and it must have been even lower concentration because the other 3 engrailed wells are coming up ok (same probe batch, but from another tube). Another issue is that there is not early gastrula like I wanted, they seem to be already have the differentiated lateral patches.

    Lunatic fringe and runx are not showing up. I exchanged AP from all wells except engrailed after 3h and then all wells before leaving at 4 °C overnight.


    Same as yesterday with NK1s and lmx coming up nicely. Exchanged AP at 10:30.


    Kept developing for 7h and stopped all wells. Nothing came up for lfng, runx or hedgehog.


    Ethanol washes for all. Added 70% glycerol with 1:10000 sytox green in hedgehog well. I want to see if embryos turn red… If not I’ll check if it is better than DAPI for these regular in situs.

  • Bruno Vellutini 19:27 on 2012/11/13 Permalink
    Tags: , , , , , , , , runx, , ,   

    Probe synthesis for Lineus et al. 

    Started a probe pcr for synthesizing the following probes of Lineus ruber, Priapulus caudatus, and Terebratalia transversa:

    Species Gene ID Probe Kit
    Lineus ruber foxa BV196 SP6
    Lineus ruber six3/6 BV199 SP6
    Lineus ruber cdx BV204 SP6
    Lineus ruber gsc BV205 T7
    Priapulus caudatus tdr BV34 T7
    Priapulus caudatus mael BV35 SP6
    Priapulus caudatus gmcl BV183 T7
    Terebratalia transversa runx2 BV166 SP6


    Run gel.


    Dna extraction.


    Transcription reaction + precipitation.



    Lr foxa 688,23
    Lr six3/6 383,21
    Lr cdx 303,11
    Lr gsc 501,31
    Pc tdr 3853,39
    Pc mael 97,89
    Pc gmcl 2287,30
    Tt runx2 345,39

  • Bruno Vellutini 12:29 on 2012/07/23 Permalink
    Tags: , , , , , , , , runx, , ,   

    PCR mostly stem/germ cell genes of Terebratalia 

    Setting up a pcr with gradient (63 +- 3 °C) at 12h:

    Gene Primers Tm mean
    Mm Six3/6 64.10
    Tt Dicer1b 61.60
    Tt Runx 63.15
    Tt Mael 65.15
    Tt Piwia 65.55
    Tt Tudor1 61.95
    Tt Ago 62.95
    Tt PL10 63.10
    Tt Pum 65.35

    Extraction and ligation overnight at 4°C, except for MAEL.


    Transformation and plating of colonies.


    PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.


    Put positive colonies to grow for minipreps.

    BV162 Terebratalia transversa Dicer1b
    BV163 Terebratalia transversa Dicer1b
    BV164 Terebratalia transversa Runx2
    BV165 Terebratalia transversa Runx2
    BV166 Terebratalia transversa Runx2
    BV167 Terebratalia transversa Tudor1
    BV168 Terebratalia transversa Argonaute
    BV169 Terebratalia transversa Argonaute
    BV170 Terebratalia transversa Argonaute
    BV171 Terebratalia transversa NK6
    BV172 Terebratalia transversa NK6
    BV173 Terebratalia transversa NK6
    BV174 Terebratalia transversa Wnt7
    BV175 Terebratalia transversa Wnt7
    BV176 Terebratalia transversa Wnt7

  • Bruno Vellutini 15:19 on 2012/05/24 Permalink
    Tags: , , , , , , runx, ,   

    Primers for more Terebratalia germline genes have arrived.

    • Tt dicer1b
    • Tt runx2
    • Tt maelstrom
    • Tt tudor1
    • Tt pl10
    • Tt ago
    • Tt pum
  • Bruno Vellutini 12:59 on 2012/05/03 Permalink
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    Cloning Tt Piwia, hh, Smo2, Nk2.2, NK5 and Mm Runx 

    Half of the plates from this batch that I had not sequenced yet. Possibly contaminated with Wnts, so I’ll need to check the bands carefully.


    Plasmid extraction and minipreps are ready for sequencing PCR.

    BV125 Terebratalia transversa Piwia
    BV126 Terebratalia transversa Piwia
    BV127 Terebratalia transversa Piwia
    BV128 Terebratalia transversa Hedgehog
    BV129 Terebratalia transversa Hedgehog
    BV130 Terebratalia transversa Hedgehog
    BV131 Terebratalia transversa Smoothened2
    BV132 Terebratalia transversa Smoothened2
    BV133 Terebratalia transversa Smoothened2
    BV134 Terebratalia transversa NK2.2
    BV135 Terebratalia transversa NK2.2
    BV136 Terebratalia transversa NK2.2
    BV137 Terebratalia transversa NK5
    BV138 Terebratalia transversa NK5
    BV139 Terebratalia transversa NK5
    BV140 Membranipora membranacea Runx
    BV141 Membranipora membranacea Runx
    BV142 Membranipora membranacea Runx


    Sequencing PCR executed and samples sent to the sequencing facility.


    Got the sequences back and they all look good and match the original sequence, except for TtPiwia, which hits Wnt1 and Wnt16. Since it is the second time I try to clone it I’ll probably re-do this gene from scratch.

  • Bruno Vellutini 18:00 on 2012/04/18 Permalink
    Tags: , , , , , runx, , ,   

    Cloning Membranipora and Terebratalia piwi, nanos, vasa, hedgehog, smoothened, runx 

    Running a PCR overnight to clone the following genes:

    Mm: Piwi1, Vasa, Runx
    Tt: Hedgehog, Smoothened, Piwia, Piwib, Vasa, Nanos.

    First time cloning Terebratalia genes.


    Membranipora bands were ok and were purified directly (and ligated), but Terebratalia were too faint:

    For this reason I ran a re-PCR for Terebratalia genes.


    For the re-PCR I diluted the PCR product of the first reaction 1:20 and used as a template for a regular PCR (without changing any parameters). The bands this time were stronger, except for Piwia. Since unwanted bands appeared I had to cut the gel, extract, and purify the DNA.

    Ligation was also set for these genes.


    Heat shock transformation for all genes.

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