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  • Bruno Vellutini 18:08 on 2012/04/19 Permalink
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    Fixation of bundles is not a good idea because the dehydration deforms the embryos a lot and it is hard to rip the membrane after re-hydration, it is tougher and less elastic (and even tighter).

    Individual embryos looked ok.

     
  • Bruno Vellutini 18:06 on 2012/04/18 Permalink
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    Rehydrate the whole egg mass to see if it was easy to take the embryos out. Kind of easy to dissect since jelly is no longer sticky and membrane is disrupted when taking the bundle off the matrix. However, embryos are slightly deformed due to dehydration.

     
  • Bruno Vellutini 18:00 on 2012/04/18 Permalink
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    Cloning Membranipora and Terebratalia piwi, nanos, vasa, hedgehog, smoothened, runx 


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    Running a PCR overnight to clone the following genes:

    Mm: Piwi1, Vasa, Runx
    Tt: Hedgehog, Smoothened, Piwia, Piwib, Vasa, Nanos.

    First time cloning Terebratalia genes.

    19/04/12

    Membranipora bands were ok and were purified directly (and ligated), but Terebratalia were too faint:

    For this reason I ran a re-PCR for Terebratalia genes.

    20/04/12

    For the re-PCR I diluted the PCR product of the first reaction 1:20 and used as a template for a regular PCR (without changing any parameters). The bands this time were stronger, except for Piwia. Since unwanted bands appeared I had to cut the gel, extract, and purify the DNA.

    Ligation was also set for these genes.

    21/04/12

    Heat shock transformation for all genes.

     
  • Bruno Vellutini 18:04 on 2012/04/17 Permalink
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    Dissected half of an egg mass keeping the egg bundles and half releasing the individual embryos. Relaxed in MgCl2 and fixed for 1h at 4°C. Normal protocol. Individual eggs look very nice and do not attach to the bottom or become disrupted. Egg bundles are a bit sticky, but after adding methanol the membrane contracts a lot, some even were disrupted; had no problem to detach the sticky ones after methanol for storage.

    Tried dissolving the sticky jelly inside the with L-Cysteine at 1%. Added first half of the volume them more. After 5 minutes the jelly was less flexible, but still sticky. Added ~1mL of 10% cysteine and the jelly dissolved almost instantly. Need to find the appropriate timing and concentration so that the embryos are not damaged. I did not relaxed before, but maybe should add MgCl2 with the cysteine. Egg bundles looked normal, maybe some eggs were sligthly disrupted, but could be from the egg mass. After methanol addition the membrane contracted as usual and embryos were kind of compressed.

     
  • Bruno Vellutini 18:00 on 2012/04/16 Permalink
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    Relaxation with MgCl2: worked fine only about 2 minutes for immersed juveniles stop moving, although some were not completely stretched (maybe because they can’t). Recovering from it took a little longer, but they were reacting every water change (about 4 times were sufficient, around 10 minutes).

    Fixation for in situ: Tried with a whole egg mass ripped to expose the egg bundles. It seemed ok (fixation and washes in Ptw) until the addition of Methanol when, apparently all the egg bundles became immersed in a jelly, maybe the inner linings dissolved with the Ptw and were condensed with the methanol? Tomorrow I’ll see how easy it is to dissect and if the eggs were not damaged.

     
  • Bruno Vellutini 18:43 on 2012/04/13 Permalink
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    Katrine/Andi Terebratalia segmentation genes, miniprep specs 


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    Since the previous sequencing for these minipreps did not work I am trying to sequence them again. These are the reads from the minipreps, they look ok.

    Gene ID ng/µL
    Tt gli 1 BV105 569.21
    Tt gli 2 BV106 755.79
    Tt gli 3 BV107 858.49
    Tt net 1 BV108 1080.2
    Tt net 2 BV109 416.45
    Tt net 3 BV110 452.18
    Tt net 3 BV111 474.99
    Tt nk6 1 BV112 1898.64
    Tt nk6 2 BV113 1217.41
    Tt nk6 3 BV114 1002.05
    Tt ptc 1 BV115 577.31
    Tt ptc 2 BV116 588.24
    Tt ptc 2 BV117 585.43
    Tt ptc 2 BV118 578.18
    Tt ptc 2 BV119 492.78
    Tt ptc 3 BV120 929.99
    Tt smo 1 BV121 631.31
    Tt smo 2 BV122 332.49
    Tt smo 3 BV123 1290.73
    Tt wnt7 1 BV124 968.39
    Tt wnt7 2 BV105 557.44
    Tt wnt7 3 BV106 2036.43
    Tt wnt7 4 BV107 554.82
    Tt wnt7 5 BV108 446.68

     
  • Bruno Vellutini 19:21 on 2012/04/12 Permalink
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    mRNA extraction for Lineus ruber 


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    Selected 1 egg mass for each time-span, dissected the bundles out, without disrupting the membrane, and kept them in gelatin coated dishes overnight. Egg bundles (and juveniles) were transfered to another dish and water exchanged once to get rid of dirt and ciliates. All bundles were put into a proper RNA eppendorf for the extraction DynaBeads protocol (link).

    Samples: 2, 4-5, 8, 11, 16 days.

    mRNA concentration: 23.8 ng/µL

    mRNA Lineus ruber

     
  • Bruno Vellutini 12:34 on 2012/04/11 Permalink  


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    Meeting with Andi to define priorities and goals. See Zim for the full content.

     
  • Bruno Vellutini 23:50 on 2012/04/01 Permalink
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    More Membranipora genes: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b 


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    Set a PCR to clone: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b

    02/04/12

    Bands with expected lengths. Purified DNA, set ligation reaction for 1h45 at RT, heatshock transformation and plated colonies with Xgal.

    03/04/12

    Plates were wetty, so colonies did not spread well… some were forming strings; but enough to pick. Picked white colonies for colony PCR:

    09/04/12

    Sequencing PCR and dropped in the SeqLab.

    BV75 Membranipora membranacea Mex3 T7
    BV76 Membranipora membranacea Fez T7
    BV77 Membranipora membranacea Fez T7
    BV78 Membranipora membranacea Fez T7
    BV79 Membranipora membranacea PL10 T7
    BV80 Membranipora membranacea PL10 T7
    BV81 Membranipora membranacea PL10 T7
    BV82 Membranipora membranacea FoxG T7
    BV83 Membranipora membranacea FoxG T7
    BV84 Membranipora membranacea FoxG T7
    BV85 Membranipora membranacea Syt1a T7
    BV86 Membranipora membranacea Syt1a T7
    BV87 Membranipora membranacea Syt1a T7
    BV88 Membranipora membranacea Syt1b T7
    BV89 Membranipora membranacea Syt1b T7
    BV90 Membranipora membranacea Syt1b T7

     
  • Bruno Vellutini 23:48 on 2012/04/01 Permalink
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    Individual eggs and bundles do not seem to… 


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    Individual eggs and bundles do not seem to be developing. Apparently more individual eggs are showing cleavage-like patterns, but I did not estimated the quantity. It does not resemble to a normal (or even unordered) cleavage.

    Some eggs inside the bundles show something more similar to a cleaving embryo, but it is hard to tell. Normally 1 or 2 eggs show this pattern while the others do not. Some eggs also look like 2 or 3 cells dividing (or fusing).

    Characteristics of the eggs have changed little since deposition.

     
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