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  • Bruno Vellutini 15:16 on 2014/06/27 Permalink
    Tags: , Owenia fusiformes   

    Set a Owenia 4D with poly-l-lysine. Strategy was to put many embryos so that at least one is well attached. Also leaving the coverslip not so close to the embryo to see if it develops further. Down part of this is that there are many embryos in the slide…

    I chose a quite stable one with animal pole view. Could not find a vegetal view…

     
  • Bruno Vellutini 15:13 on 2014/06/27 Permalink
    Tags:   

    Checked the cyphonautes cultures and they look good. There is plenty of Rhodomonas (I think they are multiplying with the light and the larvae have food in their gut. Nothing much different in the morphology, nointernal sac can be observed.

     
  • Bruno Vellutini 10:53 on 2014/06/26 Permalink
    Tags: ,   

    Set a new wild type 4D for Membranipora. Animal view, looking good.

    Folder: BCV_Mm_2014_06_26 (old Mmem_26_06_2014)

    27/06/14

    Still going well, although the late gastrula will soon not fit into the screen.

     
  • Bruno Vellutini 10:51 on 2014/06/24 Permalink
    Tags: ,   

    Started a new 4d with Membranipora. Animal view. Not so nice optics I would say, let’s see.

    Folder: BCV_Mm_2014_06_24 (old Mmem_24_06_2014)

     
  • Bruno Vellutini 13:14 on 2014/06/23 Permalink
    Tags: ,   

    After failing to keep embryos still I made poly-l-lysine slides and they stick perfectly. Just set a vegetal view recording.

    Folder: BCV_Mm_2014_06_23

    24/06/14

    Success, I stopped way ahead and it would go longer…

     
  • Bruno Vellutini 15:33 on 2014/06/22 Permalink
    Tags: ,   

    Just added another 4D recording for Membranipora. It looks ok, animal pole again.

    Folder: BCV_Mm_2014_06_22 (old Mmem_b22_06_2014)

    23/06/14

    Failed, embryo moved away.

     
  • Bruno Vellutini 15:32 on 2014/06/22 Permalink
    Tags: , , ,   

    Novocrania fluorescent in situ for mesoderm 

    Started a Novocrania in situ with engrailed and pax258, two genes that are expressed in the mesoderm. I want to find out the exact location of these genes in the coelomic pouches. And why not a double in situ with both.

    engrailed DNP pax258 DNP engrailed DNP + pax258 DIG

    23/06/14

    Added probes at 10:30.

    25/06/2014

    1st washing day. Everything normal until I was about to dilute the DNP antibody. There was 0.5 µL left and I needed 2 µL! So, passed embryos over to PTw and kept in the fridge until the new anti-dnp hrp arrives…

    01/07/2014

    Washing 5x with PBT and blocking for 1h. Adding the antibodies (anti-dnp 1:100 for engrailed and pax258 and anti-dig 1:250 for pax258 of the double) at 18h in 2mL eppendorf tubes.

    02/07/14

    Washed more than 5 times in PBT for around 10-15 minutes each. Then longer PTw washes, but not as long as 30 minutes. Started developing around 12h with TSA kit with Cy5 for the single fluorescent in situs and Cy3 for the double. Stopped the reaction with 3x 5min washes of detergent solution at 60 °C followed by 3x PTw and standard POD inactivation for the double.

    Single in situs were washed a couple of extra times in PTw and stained with 1:5000 dilution of Sytox Green for 1h30. Embryos were washed in PTw and dehydrated in Methanol. Finally embedded in Murray’s Clear.

    On the fluorescent scope the signal is not clear… there is a lot of background.

    Incubated the double with the anti-dnp antibody overnight.

     
  • Bruno Vellutini 14:28 on 2014/06/19 Permalink
    Tags: ,   

    One more Membranipora 4D recording 

    Started another recording at 1200. Animal pole view.

    File: BCV_Mm_2014_06_19 (old Mmem_19_06_14)

    Looks fine until 8 cell stage.

     
  • Bruno Vellutini 10:00 on 2014/06/19 Permalink
    Tags: , ,   

    Novocrania anomala genome 

    Started a MaSuRCA assembly with Novocrania anomala paired ends only with the following config file:

    DATA
    PE= pe 300 15  /sysdev/s9/bruno/assemblies/Novocrania_anomala/data/Nano_pe_trimmed1.fastq.gz  /sysdev/s9/bruno/assemblies/Novocrania_anomala/data/Nano_pe_trimmed2.fastq.gz
    END
    
    PARAMETERS
    #this is k-mer size for deBruijn graph values between 25 and 101 are supported, auto will compute the optimal size based on the read data and GC content
    GRAPH_KMER_SIZE=auto
    #set this to 1 for Illumina-only assemblies and to 0 if you have 1x or more long (Sanger, 454) reads, you can also set this to 0 for large data sets with high jumping clone coverage, e.g. >50x
    USE_LINKING_MATES=1
    #this parameter is useful if you have too many jumping library mates. Typically set it to 60 for bacteria and something large (300) for mammals
    #LIMIT_JUMP_COVERAGE = 60
    #these are the additional parameters to Celera Assembler.  do not worry about performance, number or processors or batch sizes -- these are computed automatically. for mammals do not set cgwErrorRate above 0.15!!!
    CA_PARAMETERS = ovlMerSize=30 cgwErrorRate=0.25 ovlMemory=8GB ovlThreads=2
    #minimum count k-mers used in error correction 1 means all k-mers are used.  one can increase to 2 if coverage >100
    KMER_COUNT_THRESHOLD = 1
    #auto-detected number of cpus to use
    NUM_THREADS= 16
    #this is mandatory jellyfish hash size
    JF_SIZE=5000000000
    #this specifies if we do (1) or do not (0) want to trim long runs of homopolymers (e.g. GGGGGGGG) from 3' read ends, use it for high GC genomes
    DO_HOMOPOLYMER_TRIM=0
    END
     
  • Bruno Vellutini 14:32 on 2014/06/18 Permalink
    Tags: , ,   

    Membranipora and U0126 inhibitor fine picked 

    Set up another U0126 inhibitor experiment. This time I’m using a fine picked and a coarse picked set of embryos. The setup is the same, but I picked only healthy 2-4 cell stage for the fine picked and a less strict sampling for the coarse picked (but still trying to fetch only good embryos.

    FSW DMSO 5 µM 7.5 µM
    1 µM 2.5 µM 10 µM 12.5 µM

    Embryos were activated at 11h, picked from 12:30-13:30  and incubated at 13:30 (fine) and 14:00 (coarse) in the facility incubator at 15 °C!

    19/06/14

    Fixed after 24h.

     
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