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  • Bruno Vellutini 18:24 on 2015/05/08 Permalink
    Tags: , , , , , , , , piwi, , ,   

    Membranipora in situ and Novocrania wnt1 

    Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.

    wnt1 dlx bmp2/4 chordin fgf
    nanos vasa piwi1 piwi2 pl10
    Na wnt1

    Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.

    09/05/15

    Added probes just after synthesis.

    11/05/15

    Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.

    12/05/15

    Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.

    13/05/15

    Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.

     
  • Bruno Vellutini 20:09 on 2015/05/06 Permalink
    Tags: , , , , piwi,   

    Probe PCR Membranipora final genes 

    Probe PCR for bmp/24, chordin, fgf8 and piwi1.

    07/05/15

    Extracted probe PCR gel.

    2015-05-07 11.35.03

    08/05/15

    Bit weird the bmp2/4, but well. Made the transcription reaction for 6:30h and put probes to precipitate.

    09/05/15

    Finish probes.

     
  • Bruno Vellutini 18:29 on 2015/04/29 Permalink
    Tags: , , , , , , piwi,   

    Membranipora cloning final genes 

    Set PCR with:

    Mm bmp2/4 Mm otx1 Mm chordin Mm fgf8/17/18
    Mm nanos Mm piwi1 Mm piwi2 Mm vasa

    30/04/15

    Ran gel:

    2015-05-01 16.23.27

    Most important genes worked: bmp2/4, chordin and fgf8. piwi1 is bonus. Extract these and set a ligation for 3h. Transformed and plated around 18h.

    01/05/15

    Set colony PCR.

    2015-05-01 16.23.36

    Put colonies to grow.

    02/05/15

    Extracted minipreps, set sequencing PCR and dropped at seq facility.

    BV378 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV379 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV380 Membranipora membranacea Chordin T7 ok + SP6
    BV381 Membranipora membranacea Chordin T7 ok + SP6
    BV382 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV383 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV384 Membranipora membranacea Piwi1 T7 ok + SP6
    BV385 Membranipora membranacea Piwi1 T7 ok + SP6

     

     
  • Bruno Vellutini 19:30 on 2012/11/16 Permalink
    Tags: brachyury, , , , , , piwi, ,   

    Lineus ruber and Membranipora in situ 

    Starting the first in situ in the nemertean Lineus ruber and trying different genes in Membranipora after establishing the ProtK timing. Selected genes are:

    L. ruber foxa six3/6 cdx gsc
    M. membranacea bra gsc piwi1 vasa

    All wells treated with 10 min in 1x protk concentration (0,01 mg/mL).

    17/11/12

    Added probes.

    19 and 20/11/12

    Washing days, normal activity. Samples stayed in blocking buffer for 1h30 (instead of 1h) due to epic foosball match.

    21/11/12

    Began developing at 11h. Twenty minutes later Mm bra cyphonautes shell were already completely blue… It seems that mucus cells in Lineus juveniles are being stained. No real signal coming up, except in six3/6, maybe. Mm piwi1 and vasa look like to be working, something is appearing. Exchanged AP once and let it at 4°C.

    22/11/12

    Changed the AP of Lineus twice. Some signal kind of visible below the epidermis, nothing in the embryos. Stopped Membranipora development.

    23/11/12

    Patterns in Lineus more visible and apparently real signal. foxa expressed in the gut region, six3/6 in the brain/anterior, cdx in the posterior tip (not in the epidermis), and gsc in the anterior mouth region. Let developing one more day at 4°C.

    25/11/12

    Stopped reaction. Background began to come up, but I could check the expression more clearly. For example, foxa is detected in the mouth region and cdx in the anus. Now what needs to be done is remove the mucus cells with cystein before fixation.

    26/11/12

    Restart development of Lineus to see if the signal get stronger in the juveniles and something appears in the embryos.

    29/11/12

    Finally stopped the development. Will do ethanol washes tomorrow.

     
  • Bruno Vellutini 14:35 on 2012/09/12 Permalink
    Tags: , , , piwi,   

    Pc gmcl and Pc piwia 

    On 05/09/12 I did a PCR for both. Only today I ran the gel and there was no band. Just setup a new reaction with lower annealing temperature (63 °C).

     
  • Bruno Vellutini 12:29 on 2012/07/23 Permalink
    Tags: , , , , , piwi, , , , , ,   

    PCR mostly stem/germ cell genes of Terebratalia 

    Setting up a pcr with gradient (63 +- 3 °C) at 12h:

    Gene Primers Tm mean
    Mm Six3/6 64.10
    Tt Dicer1b 61.60
    Tt Runx 63.15
    Tt Mael 65.15
    Tt Piwia 65.55
    Tt Tudor1 61.95
    Tt Ago 62.95
    Tt PL10 63.10
    Tt Pum 65.35

    Extraction and ligation overnight at 4°C, except for MAEL.

    24/07/12

    Transformation and plating of colonies.

    25/07/12

    PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.

    27/07/12

    Put positive colonies to grow for minipreps.

    BV162 Terebratalia transversa Dicer1b
    BV163 Terebratalia transversa Dicer1b
    BV164 Terebratalia transversa Runx2
    BV165 Terebratalia transversa Runx2
    BV166 Terebratalia transversa Runx2
    BV167 Terebratalia transversa Tudor1
    BV168 Terebratalia transversa Argonaute
    BV169 Terebratalia transversa Argonaute
    BV170 Terebratalia transversa Argonaute
    BV171 Terebratalia transversa NK6
    BV172 Terebratalia transversa NK6
    BV173 Terebratalia transversa NK6
    BV174 Terebratalia transversa Wnt7
    BV175 Terebratalia transversa Wnt7
    BV176 Terebratalia transversa Wnt7

     
  • Bruno Vellutini 18:00 on 2012/05/26 Permalink
    Tags: , , , , piwi, ,   

    Took pictures of Terebratalia in situ for germline genes vasa, nanos, piwib.

     
  • Bruno Vellutini 12:52 on 2012/05/10 Permalink
    Tags: , , , , , , piwi, , ,   

    Cloning Mm six3/6 and Tt gli, en, dlx, nk6, smo, piwia, wnt7 

    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 13:40 on 2012/05/04 Permalink
    Tags: , , , piwi, ,   

    In situ Mm Nanos and Tt Vasa, Nanos, Piwib 

    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 12:59 on 2012/05/03 Permalink
    Tags: , , , , piwi, , ,   

    Cloning Tt Piwia, hh, Smo2, Nk2.2, NK5 and Mm Runx 

    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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