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  • Bruno Vellutini 20:00 on 2013/04/30 Permalink
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    Priapulus RNA extraction of specific stages 

    Samples are neutrally buoyant with RNAlater, which makes picking a pain. I added 1x PBS with DEPC water to make the embryos sink, but still had to spin them at least 3 times at 8rpm to get all of them. Also, they stick along the wall making it hard for pipeting. In addition I spin the RNAse free tube with samples to dry out until maximum of 100µL, less when possible. Afterwards the protocol went ok.

    However, after centrifuging the isopropanol added sample a rock solid cement was fixed in the bottom of the tube. Pellet like flakes were visible with the ethanol wash and I saw them dissolving with the addition of DEPC water.

    These were the NanoDrop measurements:

    Sample ID ng/ul 260/280 260/230
    Pc oo 152,94 1,65 0,47
    Pc 1d 106,72 1,49 0,48
    Pc 2d 306,38 1,71 0,63
    Pc 3d 264,63 1,64 0,58
    Pc 4d 218,24 1,71 0,53
    Pc 5d 225,42 1,64 0,55
    Pc 6d 167,43 1,57 0,51
    Pc 7d 81,00 1,55 0,58
    Pc 9d 249,42 1,56 0,58
  • Bruno Vellutini 19:00 on 2013/04/30 Permalink

    Cystein treatment for 12/04/13 (18d) and 24/04/13 (6d) egg masses. When opening the egg mass for 18d many juveniles poped-out  and thus received the cystein treatment directly.


    Many juveniles (18d) burst because of the cystein treatment. I will need to fix this stage again. Otherwise, the earlier 6d embryos look fine.

  • Bruno Vellutini 15:54 on 2013/04/28 Permalink

    Cystein treatment for 12/04/13 (16d).


    Dissected and fixed.

  • Bruno Vellutini 19:32 on 2013/04/24 Permalink

    Cystein treatment for 10/04/13 (14d).

    Also fixed a bunch of Meara juveniles for confocal (stored in my confocal fridge box) and in situs.


    Fixed 14d.

  • Bruno Vellutini 18:11 on 2013/04/23 Permalink

    Cystein treatment for 13/04/13 (10d) and 11/04/13 (12d).


    Dissected and fixed both samples. 10d embryos were not looking so good, many seem to be loosing cells. 12d sample was great.

  • Bruno Vellutini 18:09 on 2013/04/22 Permalink

    Cystein treatment for 8d Lineus red egg mass from 14/04/13. 5 minutes in %5 cystein in FSW inside cold room. Dish washed twice with FSW and let to rock in the cold room overnight.


    Egg bundles were perfectly dissociated. I just needed to pipet a bit to release some bundles. I transfer some to a new dish and began to peel each bundle. It took me 1h30 for 1 egg mass. I fixed the embryos for 2h in the cold room + 1h at room temperature. 60% fixed for in situ and 40% for confocal.

  • Bruno Vellutini 15:39 on 2013/04/17 Permalink
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    Meara miniprep check 

    Ran a PCR to check the miniprep swap I did last time. I selected 2 sequences that will be used for in situ probes and ran a PCR with primers from both genes (boule and gus a). The correct gene should be amplified by its primer pairs and confirm the identity of the miniprep. As it can be seen in the gel I did indeed swap the labels of the miniprep, so BV226 is gus a and BV229 is boule.

    2013-04-17 15.10.07

  • Bruno Vellutini 13:51 on 2013/04/17 Permalink
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    Picked last dish of Lineus longissimus larvae with ~3 weeks old to RNALater.

  • Bruno Vellutini 16:07 on 2013/04/12 Permalink

    Had a major look into the developing embryos of Lineus sp. and here are the observations:

    0. Perfectly round fertilized [light] yellow eggs encased in bundles of 10-20 by a membrane.
    1. Some cleaving embryos are observed. Apparently one or two per bundle. I commonly saw 8 cell stage radial/holoblastic embryos.
    2. There are undivided embryos and in the first cleavage and some that look like dissociated blastomeres (80% size of original embryos). Otherwise cleavage patterns do not seem regular; embryos have blastomeres of different sizes, apparently asynchronous. For each bundle there is at least one embryo in late cleavage/morula/blastula stage which I assume to be the first embryo to undergo cleavage in the previous day.
    3. Each embryo from a bundle in different state. Some weird looking cleavages. A few blastula stages visible and maybe an initial gastrulation.
    4. Same as day 2 (and 3?), but: there are not many dividing embryos anymore, most embryos acquired a rough surface (no perfectly round eggs anymore), some blastula stages and some embryos (blastomeres?) apparently fusing.
    5. [do]
    6. Embryos look more like blastulae of different sizes and shapes. Some seem to be product of fusion of 2 embryos and some smaller ones might be dissociated blastomeres.
    7. [do]
    8. Embryos are more rounded with a smoother surface which indicates epithelia consolidation of blastula stage (probably began at 6d). I see many gastrulae, at least 1 per bundle (not all bundles are visible), with a clear blastopore on the vegetative pole. Maybe there were present since 7d, check.
    9. [do]
    10. [do]

  • Bruno Vellutini 16:46 on 2013/04/09 Permalink
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    I fixed in RNALater the second dish with Lineus longissimus larvae. I had to do an extra cleaning to get rid of super tiny bacteria/protozoa (?). So this is the sample ~1 week after spawning.

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