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  • Bruno Vellutini 12:03 on 2012/05/18 Permalink
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    Terebratalia in situ for germline genes (+Membranipora testing) 


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    First in situ using segmentation genes for Terebratalia transversa. The list is:

    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 piwi1 BV94 SP6
    Mm gata123

    Put in prehybe at 19:45.

    19/05/12

    Probes for Terebratalia genes were no good, so changing to Vasa, Nanos and Piwi:

    Tt vasa
    Tt nanos
    Tt piwi
    Tt vasa (fluo)
    Tt nanos (fluo)
    Tt piwi (fluo)
    Mm piwi1
    Mm gata

    Put the probes and let hybridizing at hybe temp.

    21/05/12

    First day of washes. Added Anti-Dig/POD (1:100) to the 3 wells of vasa, nanos, and piwi and the regular Anti-Dig/AP for the remaining; 19h and let overnight at 4° C.

    22/05/12

    Washing and started developing the regular in situ in 5 wells overnight at 4 °C.

    23/05/12

    Changes in AP Substrate: 10:30, 12:00, 14:30

    AP is becoming purpleish quite rapidly; patterns are developing accordingly fast and as expected for Terebratalia genes. Membranipora Piwi1 shows no sign of staining (although it seems to be a bit pinkish) and GATA123 is appearing, although it is not easy to identify the pattern (or if it is similar to Andi’s in situ).

    At 11:00 I started washing and then developing the fluorescent in situ. Checking under the 4D room scope the embryos show staining, but it is hard to say if the in situ worked, yet. I need to look mount them and look in the scope.

    24/05/12

    Mounted fluorescent in situ samples after staining with DAPI (1:1000) and checked in the scope. Vasa is the strongest, but still a bit weak. So next time it is better to use more probe. Nanos is there, but barely visible and Piwi is difficult to say, since the expression is widespread. Made images in the confocal and the signal seems to be there, but there is a lot of background.

    Stopped Vasa regular in situ at 10:00 and changed the substrate for the others. Changed again at 13:30. Yellow crystals were present in the Membranipora wells. Changed at 16:30 and left overnight at 4 °C.

    25/05/12

    Stopped all wells and stored in 70% glycerol.

     
  • 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 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 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.

     
  • Bruno Vellutini 17:20 on 2012/04/27 Permalink
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    Sequencing for Terebratalia segmentation genes cloned by Katrine/Andi. Just ran a sequencing PCR and sent the products to the sequencing facility.

    03/05/12

    Quickly checked the sequences today and they look bad. The only genes properly cloned were Patched and Netrin. The single Netrin sequence looks ok while the 2 Patched ones are a bit trashed.

     
  • Bruno Vellutini 18:00 on 2012/04/18 Permalink
    Tags: , , , , , , smoothened, ,   

    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 18:43 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

     
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