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  • Bruno Vellutini 17:48 on 2012/05/15 Permalink
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    Probe synthesis for Terebratalia segmentation 


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    Beginning probe synthesis for:

    Tt netrin BV110 SP6
    Tt patched BV115 SP6
    Tt hedgehog BV129 SP6
    Tt smoothened2 BV131 SP6
    Tt nk2.2 BV135 SP6
    Tt nk5 BV139 SP6
    Mm ago2 BV25 SP6
    Mm mago nashi BV27 T7
    Mm germ cell less BV30 T7

    Run the PCR and letting overnight to extract tomorrow.

    16/05/12

    Ran gel and extracted the bands. Looking ok:

    18/05/12

    Started probe synthesis reaction at 10:40. Precipitated around 16h.

    19/05/12

    Terebratalia probes had no pellet (but in fact, I don’t know if I centrifuged it). Membranipora using SP6 had a small pellet and the two T7 ones had a huge pellet. Specs are:

    Gene ng/µL
    Mm Ago2 293.92
    Mm Magoh 3806.73
    Mm GMCL 3147.70

    Diluted to 50 ng/µL and stored at -20 °C.

    21/05/12

    Re-doing the transcription reaction for Terebratalia segmentation genes. Started at 11h, but only put at 37°C at 13h (my bad…). Precipitated at 17h30 using only 5 µL of Lithium Chloride. Could not see any pellet…

    22/05/12

    After centrifuging a thin pellet appeared in the bottom of the tubes. I used Low yield, but enough for making probes:

    Gene ng/µL
    Tt netrin 120.56
    Tt patched 174.64
    Tt hedgehog 120.93
    Tt smo2 107.95
    Tt nk2.2 211.83
    Tt nk5 221.16

    Diluted to 50 ng/µL and stored in the freezer.

     
  • Bruno Vellutini 13:09 on 2012/05/14 Permalink
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    Fixation of 11d old embryos of L. ruber (L: 03/05/12) for in situ and confocal. Common procedure without using cysteine. Also took a few pictures. There were some apparently Desor’s larval stage… or at least some ciliated funny looking embryos.

     
  • Bruno Vellutini 17:07 on 2012/05/11 Permalink
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    Fixation of 41d old juveniles (L: 01/04/12) for in situ and confocal. Relaxation with MgCl2 worked quite well. In less then 8 minutes all worms were relaxed.

     
  • Bruno Vellutini 16:45 on 2012/05/10 Permalink
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    Fixation of 7 d old L. ruber for confocal and in situ. Bissected the halves of egg masses from previous fixation and removed the outer case. Instead of treating with cystein I simply used the large plastic pipet to dissociate the eggs from the jelly. It worked quite well, even though the jelly would stick in the pipet.

    Eggs were release from the bundle with needles and fixed with the standard protocol for in situ and confocal.

     
  • Bruno Vellutini 12:52 on 2012/05/10 Permalink
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    Cloning Mm six3/6 and Tt gli, en, dlx, nk6, smo, piwia, wnt7 


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    Started a regular PCR for (re-doing this genes from scratch):

    Mm: six3/6
    Tt: piwia, engrailed, distal-less, gli, nk6, smoothened, wnt7

    Tt bands were not good, so trying a re-PCR overnight.

    11/05/12

    In the end the re-PCR did not worked well, so I purified the DNA directly for  Mm six3/6 and Tt piwia, nk6, and smoothened. Ligation was set for these and let over the weekend.

    14/05/12

    Heatshock transformation and plating at 18h.

    15/05/12

    Colony PCR and selected colonies to grow at 17h30. Tt gli is being grown from Katrine’s plate. Bands were kind of variable… do not like this; let’s see if it is contamination.

    BV143 Membranipora membranacea Six3/6 T7
    BV144 Membranipora membranacea Six3/6 T7
    BV145 Membranipora membranacea Six3/6 T7
    BV146 Terebratalia transversa Piwia T7
    BV147 Terebratalia transversa Piwia T7
    BV148 Terebratalia transversa Piwia T7
    BV149 Terebratalia transversa Piwia T7
    BV150 Terebratalia transversa NK6 T7
    BV151 Terebratalia transversa NK6 T7
    BV152 Terebratalia transversa NK6 T7
    BV153 Terebratalia transversa Smoothened T7
    BV154 Terebratalia transversa Smoothened T7
    BV155 Terebratalia transversa Smoothened T7
    BV156 Terebratalia transversa Gli T7
    BV157 Terebratalia transversa Gli T7
    BV158 Terebratalia transversa Gli T7

    18/05/12

    Set a sequencing PCR at 11:40; delivered at 19:45.

    23/05/12

    Many Wnts mixed with the proper genes again… Interestingly, Gli samples, which I prepared the minipreps directly from Katrine’s plate did not have Wnts. This indicates that the contamination event happened afterwards, possibly during Henrike’s stay. It seems that something in the ligation kit is the culprit.

     
  • Bruno Vellutini 18:51 on 2012/05/08 Permalink
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    Picked juveniles from original dishes (dissected the ones that were still inside the egg masses) and transferred them to clean dishes. The morphology looks quite similar within similar size ranges. There are many small-sized worms that look a bit different, more round for example.

    I took some pictures and made some videos of the juveniles moving around and leaving the egg mass.

     
  • Bruno Vellutini 15:00 on 2012/05/05 Permalink
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    Fixation of 2 d old L. ruber embryos for in situ and confocal. I dissected half of 2 egg masses for fixation and keeping the respective halves to follow further development. Fixed using the established protocol for fixing nemertean embryos.

    1. Remove outer case of the egg mass keeping the jelly with the bundles attached.
    2. Immerse the jelly+bundles in 5% cysteine freshly made for 5 min rocking (can be increased if jelly is still sticky, but not too long since cystein can damage the embryos).
    3. Use a large pipet to pipet up and down to release the bundles from the jelly.
    4. Using needles remove the remaining bundles from the jelly and wash 2x in FSW.
    5. Open individual bundles to release the embryos.
    6. Proceed with regular fixation protocol for in situ or confocal.

    Took me about 1h30 to reach step 6… Wondering if this could be done faster.

     
  • Bruno Vellutini 13:40 on 2012/05/04 Permalink
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    In situ Mm Nanos and Tt Vasa, Nanos, Piwib 


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    Trying 2 samples of late cyphonautes of Membranipora, one without proteinase-k treatment and one with 30s half-concentrated dose. Also, samples of all stages of Terebratalia using the probes for Vasa, Nanos, and Piwib.

    Mm Nanos Mm Nanos Tt Vasa Tt Nanos Tt Piwib
    no prot-k 30s half-conc prot-k 10min prot-k 10min prot-k 10min prot-k

    Membranipora embryos/larvae take 1 min to sink. When trying to do 30s of prot-k I managed to start removing the solution at 15s, but only finished and added glycine around 45s. I added a double dose of glycine to dilute the prot-k well and quickly did the second wash (also with double amount). Membranipora embryos treated with prot-k were fixed for 1h30 while Terebratalia for 1h.

    05/05/12

    Added the probes to the wells and let it hybridize over the weekend.

    07/05/12

    First day of washes. Membranipora embryos look ok (not dissolving) and Terebratalia became a bit more transparent, but also look integer. Put the antibody at 18:30, incubating overnight rocking at 4 °C.

    08/05/12

    Washes with PBT and PTw ran as expected. Letting overnight at 4 °C. Embryos are still there and apparently no differences between prot-k treatment and control of Membranipora.

    09/05/12

    Developing from 11h.

    30min: Apparently nothing on Membranipora and Terebratalia Nanos and Piwib. Terebratalia Vasa is already showing the expression, a bit more broadly expressed in gastrula stages and restricted to a ring in the mid-lobe of larvae.
    60min: Nothing on Mm. Tt Nanos and Piwib started to develop a faint staining in the archenteron region of the gastrula. Tt Vasa continues to show a strong staining pattern.
    90min: Same.
    3h: TtVasa looks stronger and TtNanos starting to appear more clearly.
    5h: Same pattern. Changing the AP substrate and leaving at 4 °C overnight.

    10/05/12

    Stopped Tt Vasa at 10h (following PTw washes). Changed the AP substrate for the others. Signal is stronger in Nanos and Piwib, better revealing the pattern. Apparently nothing is coming up in Mm, although I saw some unspecific bilateral staining appearing.

    Changed AP substrate at 10h, 14h, and 18h. Leaving overnight at 4°C.

    11/05/12

    Forgot to filter the AP substrate, so wells were full of yellow crystals in the bottom and covering the samples… :( I stopped the reaction and did the Ethanol washes for all wells. Crystals were mostly dissolved, but wells became dirty. After ethanol washes it was all good again. Stored in the fridge with 70% glycerol in PTw.

    14/05/12

    Mounted Membranipora samples with and without proteinase-k. Both look similar and show no expression of Nanos in the expected region. So it seems that the in situ did not work again. At least the embryos made it to the end of the protocol in one piece.

     
  • Bruno Vellutini 19:00 on 2012/05/03 Permalink  


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    Observed that the longer and thinner nemerteans triaged on Tuesday are fragmenting. I’ll keep the fragments to see if they will regenerate.

     
  • Bruno Vellutini 12:59 on 2012/05/03 Permalink
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    Cloning Tt Piwia, hh, Smo2, Nk2.2, NK5 and Mm Runx 


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    Half of the plates from this batch that I had not sequenced yet. Possibly contaminated with Wnts, so I’ll need to check the bands carefully.

    04/05/12

    Plasmid extraction and minipreps are ready for sequencing PCR.

    BV125 Terebratalia transversa Piwia
    BV126 Terebratalia transversa Piwia
    BV127 Terebratalia transversa Piwia
    BV128 Terebratalia transversa Hedgehog
    BV129 Terebratalia transversa Hedgehog
    BV130 Terebratalia transversa Hedgehog
    BV131 Terebratalia transversa Smoothened2
    BV132 Terebratalia transversa Smoothened2
    BV133 Terebratalia transversa Smoothened2
    BV134 Terebratalia transversa NK2.2
    BV135 Terebratalia transversa NK2.2
    BV136 Terebratalia transversa NK2.2
    BV137 Terebratalia transversa NK5
    BV138 Terebratalia transversa NK5
    BV139 Terebratalia transversa NK5
    BV140 Membranipora membranacea Runx
    BV141 Membranipora membranacea Runx
    BV142 Membranipora membranacea Runx

    05/05/12

    Sequencing PCR executed and samples sent to the sequencing facility.

    08/05/12

    Got the sequences back and they all look good and match the original sequence, except for TtPiwia, which hits Wnt1 and Wnt16. Since it is the second time I try to clone it I’ll probably re-do this gene from scratch.

     
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