Updates from October, 2012 Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 14:09 on 2012/10/31 Permalink
    Tags: funding,   

    Submitted EDEN proposal 

    Just submitted my proposal to EDEN to go to Casey’s lab to process RNA-Seq data of different developmental stages of Priapulus. Compiled file sent today.

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

     
  • Bruno Vellutini 18:44 on 2012/10/26 Permalink
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    Membranipora in situ Syt1a and Nanos 

    In situ with Membranipora for testing the newly fixed material with glutaraldehyde. I’m using 4 conditions and 2 genes:

    ProtK Time Gene Gene
    0.5x 10 min Syt1a Nanos
    0.5x 20 min Syt1a Nanos
    1x 20 min Syt1a Nanos
    2x 20 min Syt1a Nanos

    31/10/12

    Developing the in situ. Wells with nanos already showing the background all around with no difference from treatments. Synaptotagmin looks more promising, no background still. First well seems to have a slight purpleish regions, but others are plain white. Exchanged the AP twice and let it overnight in cold room.

    01/11/12

    Nanos is completely background. On syt1a I think there is signal in the last two treatments (1x and 2x). The lighter treatments seem to have some background. I exchanged the AP twice and will stop the reaction today.

    02/11/12

    It seems that synaptotagmin works.

     
  • Bruno Vellutini 18:46 on 2012/10/25 Permalink
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    ProteinaseK test for Membranipora 

    Tested Membranipora to see how do I have to treat them in ProteinaseK during the in situ. I put 5 larvae in 0.005 mg/mL (half-concentrated) and waited 1h checking every 5 minutes. Nothing changed. I added +2.5 µL of ProtK (up to 0.01 mg/mL) and waited 30 min more and nothing changed. So I left the embryos overnight and in the following morning only the shell was left, soft tissues were dissolved.

     
  • Bruno Vellutini 18:49 on 2012/10/12 Permalink
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    Membranipora in situ with new material 

    Starting an in situ with the newly fixed material of Membranipora. I took 200 µL from X, X, X, X days and mixed. Since samples were fixed with 3.7% PFA + 0.2% Gluta I am testing the time in proteinase K for 2 genes, Synaptotagmin and Nanos (a strong one and a known one), with 30s and 1:30s.

    30s 1m30s
    Mm gene Syt1a Syt1a
    Mm gene Nanos Nanos

    17/10/12

    Started developing at 11h30. By night a weak background began to appear in the shell.

    18/10/12

    Change AP at 10h. Background became stronger and apparently no real signal neither in syt or nos. Some larvae in the syt wells do not have much background. Crystals appeared in the afternoon… Kept developing at 4 °C.

    21/10/12

    Stopped developing.

    24/10/12

    Checked under microscope… only background it seems.

     
  • Bruno Vellutini 14:21 on 2012/10/04 Permalink
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    I started Lineus ruber transcriptome assembly with Casey’s pipeline AGALMA. Since velvet was not compiled with multithread capabilities and oases is not multithreaded it was taking too long (24h). I then stopped it and managed to start an assembly with CLC. With the following parameters:

    Automatic word size = ON
    Automatic bubble size = ON
    Minimun contig length = 170
    Perform scaffolding = OFF
    Map reads back = ON
      mismatch = 2
      insertion = 3
      deletion = 3
      length = 0.5
      similarity = 0.8
    Global alignment = OFF
    Update contigs = ON
    Creat list of un-mapped = OFF
    

    It started at 21h30 and finished ~06h00 in the next morning.

    results

     
  • Bruno Vellutini 18:23 on 2012/10/01 Permalink
    Tags:   

    Fixed adult colonies for some in situs.

    Relaxed with MgCl2 for 20 min, they immediatly go out of the cystid. However, next time I’ll let them go out with the light and then add the magnesium to try to relax a higher porcentage of zooids; not all came out.

    I’m fixing in 3.7% overnight.

     
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