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  • Bruno Vellutini 12:44 on 2013/02/13 Permalink
    Tags: caudal, , , , ,   

    Probe synthesis for old and new Terebratalia segmentation genes 


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    Transcription reaction set for at 10h30 and precipitation at 17h00:

    ID GENE ENZYME UTP ng/µL
    BV208 en SP6 DIG 135
    BV208 en SP6 DNP 55
    BV209 dlx SP6 DIG 357
    BV211 wnt4 SP6 DIG 351
    BV218 wnt11 SP6 DIG 1234
    BV210 cdx T7 DIG 1450
    BV212 wnt8 T7 DIG 129
    BV213 eve T7 DIG 2269

    14/02/13

    Pellet washing and specs taken, see above. en was the least concentrated one, specially with DNP utp. Will need to re-do it to get more probe.

     
  • Bruno Vellutini 17:17 on 2013/02/07 Permalink
    Tags: caudal, , , , , , ,   

    Transformation of plasmids for Terebratalia new probes 


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    Some of the genes cloned by Yale and Andi will be used for double in situs. We have no probes and there is a limited amount of plasmid solution. So I’m transforming the plasmids again to make more minipreps (and then probes). The genes are:

    Gene ID Kit
    en TTR54 SP6
    dlx Tt-dlx-L2 SP6
    cdx TTR16 T7
    wnt4 TTR164 SP6
    wnt8 TTR104 T7
    eve Tt-eve-L2 T7

    I used 1 µL of plasmids for the transformation and diluted the bacteria solution prior to plating by 1:100 (1µL bacteria: 99µL LB at 37°C). I then plated 50 µL of the 1:100 solution at 16h30.

    08/02/13

    Perfect number of colonies!

    2013-02-08 17.42.06

    09/02/13

    T7
    en BV167
    dlx BV168
    cdx BV169
    wnt4 BV170
    wnt8 BV171
    eve BV172

    10/02/13

    Extracted the plasmids and started the probe PCR.

    11/02/13

    Probe PCR extracted.

    2013-02-11 15.07.35

     
  • Bruno Vellutini 19:30 on 2012/11/16 Permalink
    Tags: brachyury, caudal, , , , , , ,   

    Lineus ruber and Membranipora in situ 


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    Starting the first in situ in the nemertean Lineus ruber and trying different genes in Membranipora after establishing the ProtK timing. Selected genes are:

    L. ruber foxa six3/6 cdx gsc
    M. membranacea bra gsc piwi1 vasa

    All wells treated with 10 min in 1x protk concentration (0,01 mg/mL).

    17/11/12

    Added probes.

    19 and 20/11/12

    Washing days, normal activity. Samples stayed in blocking buffer for 1h30 (instead of 1h) due to epic foosball match.

    21/11/12

    Began developing at 11h. Twenty minutes later Mm bra cyphonautes shell were already completely blue… It seems that mucus cells in Lineus juveniles are being stained. No real signal coming up, except in six3/6, maybe. Mm piwi1 and vasa look like to be working, something is appearing. Exchanged AP once and let it at 4°C.

    22/11/12

    Changed the AP of Lineus twice. Some signal kind of visible below the epidermis, nothing in the embryos. Stopped Membranipora development.

    23/11/12

    Patterns in Lineus more visible and apparently real signal. foxa expressed in the gut region, six3/6 in the brain/anterior, cdx in the posterior tip (not in the epidermis), and gsc in the anterior mouth region. Let developing one more day at 4°C.

    25/11/12

    Stopped reaction. Background began to come up, but I could check the expression more clearly. For example, foxa is detected in the mouth region and cdx in the anus. Now what needs to be done is remove the mucus cells with cystein before fixation.

    26/11/12

    Restart development of Lineus to see if the signal get stronger in the juveniles and something appears in the embryos.

    29/11/12

    Finally stopped the development. Will do ethanol washes tomorrow.

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

    Probe synthesis for Lineus et al. 


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    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

    13/11/12

    Run gel.

    14/11/12

    Dna extraction.

    15/11/12

    Transcription reaction + precipitation.

    16/11/12

    Specs:

    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 16:14 on 2012/11/05 Permalink
    Tags: caudal, , , , , ,   

    Some cloning of Lineus and Priapulus 


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    Cloning of Pc gmcl, Lr caudal, Lr goosecoid, Lr foxq2, Lr fgf 8/17/18. Running the PCR with 60 °C for annealing, adjusted to primers Tm.

    06/11/12

    Running gel:

    I cut caudal and goosecoid bands. Extracted, ligated/transformed.

    09/11/12

    Colony PCR:

    10/11/12

    Minipreps for cloning:

    Colony T7
    cdx 6 BV202
    cdx 7 BV203
    cdx 8 BV204
    gsc 6 BV205
    gsc 7 BV206
    gsc 8 BV207

     
  • Bruno Vellutini 19:01 on 2012/10/29 Permalink
    Tags: caudal, , , , , , ,   

    Lineus ruber first cloning 


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    Trying to clone 6 genes: six 3/6, caudal, goosecoid, foxa, foxq2, and fgf8/17/18. PCR was not so successful, only foxa and six3/6 worked:

    30/10/12

    Set ligation.

    31/10/12

    Transformed and plated.

    01/11/12

    Colony PCR for both genes:

    03/11/12

    Minipreps.

    Colony T7
    foxa 4 BV196
    foxa 5 BV197
    foxa 6 BV198
    six3/6 6 BV199
    six3/6 7 BV200
    six3/6 8 BV201

     
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