Tagged: Lineus longissimus Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 11:41 on 2014/06/05 Permalink
    Tags: , Lineus longissimus,   

    RNA extraction of Lineus longissimus embryos and juveniles 

    Starting extraction with snap-frozen samples of different stages of Lineus longissimus with the TriReagent protocol.

    # stage date ng/µL 260/280 260/230 total ng
    1 late cleavage 0d 02/04/14 204.0 1.89 1.83 4794.0
    2 blastula 1d 03/04/14 189.2 1.88 1.98 4446,2
    3 gastrula 2d 04/04/14 182.3 1.88 2.15 4284,05
    4 pilidium 3d 05/04/14 319.6 1.92 1.75 7510,6
    5 pilidium 5d 07/04/14 158.8 1.85 1.27 3731,8
    6 juvenile 2 months 12/05/14 50.3 1.77 1.03 1182,05
    7 juveniles 3 months 13/06/14 41

    Since the samples of the juveniles look so bad I think I’ll do another extraction.

     
  • Bruno Vellutini 11:26 on 2014/06/05 Permalink
    Tags: Lineus longissimus   

    Fixing Lineus longissimus juveniles 

    Picked a batch of juveniles of the nemertean Lineus longissimus for in situ and immuno staining fixation.

    1. Relaxed for 1h in 7.4% MgCl2.
    2. Separated 1/4 of the worms for immuno on a tube.
    3. Just before adding the cystein I noticed that the worms were beginning to dissolve. So I only added 2% cystein in 7.4% MgCl2 for 30 s and immediately exchanged the solution for 4% FA. There are still some integer worms, so it should be ok. They should not be relaxed for more than 1h.
    4. Fixation for 1h at RT.
    5. Washed with PTw and then methanol for in situ samples.

    Relaxation seem to work, but many worms were not fully extended. The posterior end was curved ventrally.

     
  • Bruno Vellutini 00:06 on 2014/05/13 Permalink
    Tags: Lineus longissimus   

    Washed multiple times Lineus longissimus juveniles for RNA extraction. Put them into a RNA tube and wahsed and centrifuged more than 3 times with uvfiltered sea water. Then snapfreeze. They were around 40 days old.

     
  • Bruno Vellutini 16:12 on 2014/05/08 Permalink
    Tags: Lineus longissimus   

    Washed Lineus longissimus larvae. Many larvae in the more advanced jar at 19 °C had already settled. I removed the swimming larvae by filtering the water until there was only a finger of water left. Then I rinsed the bottom to release the juveniles and transferred everything to a glass bowl.

    Juveniles look happy. I washed and cleaned the bowl as much as possible, but the juveniles stick to the bottom and to the pipets, so it is a bit hard. I transferred most of the worms to a clean bowl and kept the dirty as well in the incubator at 14 °C. I’ll separate many for RNAlater, a few for immunos and the rest to raise until adulthood.

    The other jar in fritzen room does not look so good. I’ll have a look at it next week. The jar from the incubator has many larvae, but they have the least advanced rudiment worm inside.

    I forgot to add food. I will add tomorrow.

     
  • Bruno Vellutini 20:17 on 2014/04/29 Permalink
    Tags: Lineus longissimus   

    Washed and fed the larvae of Lineus longissimus. In the old jar the pilidium larvae have a well-formed juvenile with eyes, it will soon settle. In the second jar the juveniles are visible, but in a much earlier stage.

    20140429_210448

    I will let these metamorphose and fix this pilidium with well-developed juveniles after nice from the second jar.

     
  • Bruno Vellutini 19:53 on 2014/04/15 Permalink
    Tags: Lineus longissimus   

    I took a look at Lineus longissimus pilidium larvae and observed the following.

    The larvae from the culture with stirring at 19 °C look the best. They are larger with food and more responsive. This can be due to the higher temperature. But I think stirring keeps the algae floating and optimizes the feeding. On the two large jars in the incubator there was a smoodge of Rhodomonas on the bottom while the bottom of the fritzen room was relatively clean.

    Larvae on the bowl looked ok at the naked eye (swimming and so), but at the scope they are small and with contracted lobes. Larvae on the beaker are better, but not as good as the large jars. The large jars in the incubator have happy larva, but they are smaller and seem to have gotten less food.

    I tried pipetting the bottom before sieving the beaker, but it was not effective. And maybe the larvae will try to eat the dirt! So for now it seems better to keep the bottom untouched.

    I used the filter of 50µm from FHL and the small or larger rubber tube (much faster) to remove half of the volume of the water. Replaced with UV filtered water at RT from our facility,

     
  • Bruno Vellutini 18:47 on 2014/04/10 Permalink
    Tags: Lineus longissimus   

    Pilidium larvae of Lineus longissimus look wonderful and happy. Annie changed 1/3 of the water today and added more food. There was algae sticking to the bottom, so maybe next time it needs to be scrubbed so that it gets exchanged.

    I’ll do the next cleaning on Monday.

     
  • Bruno Vellutini 19:55 on 2014/04/07 Permalink
    Tags: Lineus longissimus   

    We decided to try to raise the larvae longer so that juveniles genes are also expressed. Thus, we have set up a stirring pad on a 3L plastic jar at Fritzen’s room, which is around 19 °C. We will probably change this setup to something different tomorrow.

    08/04/14

    We diluted cultures to 1 larva per mL in 4L clear plastic jars and put them in the incubator at 14 °C with some light bubbling. There are 2 4L jars plus 1 1L beaker. We put an additional 4L jar on Fritzen room with stirring to check for any differences.

    Annie checked the larva and they had algae in their gut!

     
  • Bruno Vellutini 13:38 on 2014/04/02 Permalink
    Tags: Lineus longissimus   

    Lineus longissimus spawned! 

    Lineus longissimus spawned today, probably early morning. Embryos were already at mid/late cleavage stage. We concentrated them and did some washes with UVFSW. I’ll start snap freezing for RNA extraction today and once per day. We need to get a transcriptome out of it.

    Date Snap-freeze sample
    1 02/04/14 0d Llon late cleavage
    2 03/04/14 1d Llon blastula (date in the tube is wrong… says 04/04)
    3 04/04/14 2d Llon late gastrula
    4 05/04/14 3d Llon pilidium
    5 07/04/14 5d Llon pilidium
    6 12/05/14 2 month juvenile

     

     
  • Bruno Vellutini 20:37 on 2013/05/04 Permalink
    Tags: Lineus longissimus, ,   

    RNA extraction for 2nd series of Priapulus stages and L. longissimus 

    Starting an extraction for another series of Priapulus stages. Using Phase Gel Lock tubes, LPA carrier and overnight precipitation.

    Embryos were at the bottom so it was a little easier to pick, but in the end I had to spin them many times to dry out the collecting tube to 100 µL. Another problem was using 1 mL for separating the phases in the gel lock tubes. They only support 750 µL. For this reason the gel was at the top in most tubes. So I collected the aqueous phase with Chema’s help and mixed an equal volume of chloroform to take the phenol out. Only after this we added the carrier (1 µL) and let it precipitate overnight.

    05/05/13

    Some tubes had a transparent bottom phase. After spinning this unknown droplet trapped the pellet, nothing changed after the ethanol wash. When I added 22 µL of DEPC water the pellet and droplet seem to have dissolved. In the tubes where there was a clear and nice pellet a solid case remained where the pellet was. These are the results of the NanoDrop:

    Sample ID ng/ul 260/280 260/230
    Pc2 oocytes 106,74 1,78 0,41
    Pc2 1d 186,70 1,95 0,38
    Pc2 2d 236,72 2,18 0,50
    Pc2 3d 246,45 2,04 0,50
    Pc2 4d 834,65 1,93 0,43
    Pc2 5d 215,32 2,13 0,43
    Pc2 6d 112,33 1,68 0,39
    Pc2 7d 303,38 1,85 1,37
    Pc2 9d 283,21 2,04 0,55
    Pc2 12d 390,20 1,92 0,52
    Pc2 15d 485,06 1,94 0,96
    Pc2 2nd larvae 131,70 1,74 0,40
    Llon mixed 288,17 1,85 0,59

    Candidates for re-extraction are: oocytes, 7d, 2nd larvae

     
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