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  • Bruno Vellutini 15:22 on 2012/05/25 Permalink
    Tags: , , , netrin, , , , ,   

    In situ segmentation genes Terebratalia 

    Starting an in situ for segmentation genes in Terebratalia and repeating the fluorescent in situ for Vasa and Nanos, trying to reduce the background. The plate looks like this:

    netrin patched hedgehog vasa
    smo2 nk2.2 nk5 nanos

    For vasa and nanos I will use a lower concentration of the antibody, 1:500 (last time it was 1:100) — effort to reduce background. For nanos I will also double the amount of probe to get a stronger signal.

    Samples put in prehybe at 17h:00.

    26/05/12

    Added regular probes at 1 ng/µL, except for nanos which was 2 ng/µL (trying to increase the signal/noise ratio for fluorescent in situ).

    28/05/12

    Washes. Added regular Anti-Dig/AP to segmentation genes (1:5000) and Anti-Dig/POD (1:250) to vasa and nanos (in an attempt to lower the background noise — used 1:100 previously).

    29/05/12

    Regular washes with PBT and PTw.

    30/05/12

    Started developing segmentation genes of Terebratalia at 11:30. For the fluorescent in situ I developed normally for 1h30 and then stopped the reaction with a detergent solution used by (10.1038/nature10838) to reduce the background; 3 washes of 20 min at 62 °C. The solution:

    [list of chemicals]

    I checked the slides in the fluorescent lamp and did not see any staining. It is also not so clear if there is less background. I guess I need to check in the confocal.

    The segmentation genes began to appear. Netrin is the strongest one and the pattern is interesting. I notice that the “ring” background might be covering the whole anterior of the pedicle lobe. Other genes are still weak to say something.

    Changed the AP substrate at 16:30 and let it developing at 4 °C.

    31/05/12

    Patterns are more clear today. Changed the substrate 3 times (10:15, 12:30, 16:00) and let it overnight at 4 °C. Crystal began to appear after lunch and are already covering the whole bottom of the well.

    01/06 – 04/06

    Stopped netrin, nk2.2, and nk5. Developed others until 04/06. Many crystals forming…

    Bibliography

     
  • Bruno Vellutini 12:03 on 2012/05/18 Permalink
    Tags: , , netrin, , , ,   

    Terebratalia in situ for germline genes (+Membranipora testing) 

    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
    Tags: , , , , , netrin, , , , ,   

    Probe synthesis for Terebratalia segmentation 

    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 17:20 on 2012/04/27 Permalink
    Tags: , netrin, , , , , , ,   

    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 18:43 on 2012/04/13 Permalink
    Tags: , , , netrin, , , , , ,   

    Katrine/Andi Terebratalia segmentation genes, miniprep specs 

    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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