Tagged: goosecoid Toggle Comment Threads | Keyboard Shortcuts

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

    Lineus ruber and Membranipora in situ 

    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: , , , goosecoid, , , , , , , ,   

    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

    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: , , , , goosecoid, ,   

    Some cloning of Lineus and Priapulus 

    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: , , , , goosecoid, , ,   

    Lineus ruber first cloning 

    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

     
c
Compose new post
j
Next post/Next comment
k
Previous post/Previous comment
r
Reply
e
Edit
o
Show/Hide comments
t
Go to top
l
Go to login
h
Show/Hide help
shift + esc
Cancel