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  • Bruno Vellutini 18:24 on 2015/05/08 Permalink
    Tags: , , distal-less, , , , , , , , ,   

    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 18:45 on 2015/03/30 Permalink
    Tags: distal-less, , , , , , , ,   

    Membranipora wnt in situ 

    First new try with bryozoan in situ. Separate wells for early (until extended gastrula) and late stages (early cyphonautes and later cyphonautes).

    early stages 1x nk2.1 1x wnt1 1x wnt5 1x wnt7 1x wnt8
    late stages 0.1x nk2.1 0.1x wnt1 0.1x wnt5 0.1x wnt7 0.1x wnt8
    early stages 1x dlx 1x gbx 1x pax258 1x nanos 1x vasa
    late stages 0.1x dlx 0.1x gbx 0.1x pax258 0.1x nanos 0.1x vasa

    After protk step (10 min) embryos were suspended in glycine (added 800 µL) and did not sink. I added 600 µL of glycine for the second wash. The whole glycine steps lasted for 15 min. The remaining washes were a bit longer than 5 min, but I could control it better.

    I noticed that the embryonic membrane dissolves with protk.

    Let overnight in pre hybe.

    31/03/2015

    Diluted and added probes to in situ (first 0.1x then 1x). Left oven set to 63 °C (62 in the electronic thermometer and 63 in the mercury thermometer).

    02/04/2015

    First hybe wash I removed 1x probes first, filled with warm hybe wash and then removed 0.1x probes and added warm hybe.

    • 1x 10min hybe wash
    • 1x 50min hybe wash
    • 1x 30min 75% hybe
    • 1x 30min 50% hybe
    • 1x 30min 25% hybe
    • 1x 30min 2x SSC
    • 3x20min 0.2x SSC
    • 1x 10min 75% 0.2x SSC
    • 1x 10min 50% 0.2x SSC
    • 1x 10min 25% 0.2x SSC
    • 1x 10min PTw.
    • 4×10-15min washes in PBT (total 45min)
    • 1h in 1x blocking buffer
    • Overnight at 4°C with anti-dig-ap

    03/04/2015

    • 3x 5min PBT
    • 4x 15min PBT
    • 5x 30min PTw

    Developed in situ. A significant amount of pinkish background came up in my probes. wnt5 was fast, also dlx. Others had different levels of background in addition to the real signal, except Chema’s probe for NK2.1. Thus, this background is likely due to probe design or probe synthesis. Here are the lengths of the clones used to synthesize the probes:

    gene nk2.1 wnt1 wnt5 wnt7 wnt8 dlx gbx pax2/5/8 nanos vasa
    bp 1113 1038 1073 869 886 823 888 902 868 812

    Length of the probe can be the issue but it does not quite explain the rapid background coming up. Longer probes should result in a better signal to noise ratio because the signal comes up faster and not because the background comes up slower. It can also be something related to the probe synthesis process. But what? I usually get high yields and good peak ratios. Check the numbers to see!

    Ah, one obvious thing is the probe concentration. NK2.1 probe is probably more diluted than the others since it is its second or third use. Thus, the next in situ should be lower concentration or higher temperature.

    04/04/15

    Continued developing wnt7 and gbx and stopped them after 3h. Ethanol washes with 20min in 100% and let in PTw overnight at 4° C because glycerol was not ready.

    05/04/15

    Putting them in 70% glycerol with DAPI. Will put apart some of the NK2.1 embryos to do a MAPK immuno staining.

     
  • Bruno Vellutini 16:03 on 2014/06/06 Permalink
    Tags: distal-less, , , , , ,   

    Brachiopod in situ 

    In situ for additional Terebratalia frizzleds and repeating some Novocrania for better pictures.

    Nano engrailed wnt1 wnt5 dlx pax258
    Ttra frizzled 5/8 frizzled 7 dcr1b tdr1 ago

    Started in situ with standard procedures. 8 min protk for Novocrania and 10 min for Terebratalia.

    07/06/14

    Added probes, embryos look good.

    09/06/14

    Washing day. Everything went as usual and put antibody around 18h.

    10/06/14

    Washed with PBT several times berween 10-15 min. Then PTw 30 min washes. Started developing.

    Stopped Nano dlx after 2h30min because it was done, as well as Ttra fz5/8. The rest continued developing. fz7 got pinkish really fast

    11/06/14

    fz7 and ago got really dark. I don’t know if it is background or signal, even though it looks like there is real signal within the background. I stopped them as well as engrailed and pax258, which look better than the last in situ.

    12/06/14

    Stopped wnt1, wnt5 dcr1b, tdr1. Novocrania samples are looking dark ugly already…

    13/06/14

    Trying to clear out some of the background from fz7 and ago… letting in ethanol for many hours.

     
  • Bruno Vellutini 10:11 on 2014/05/08 Permalink
    Tags: , , distal-less, , ,   

    Probe synthesis Ttra frizzleds Nano dlx and more 

    Gene Miniprep Antisense Enzyme ng/µL
    Nano dlx BV285 SP6  159
    Ttra fz5-8 BV288 SP6  366
    Ttra fz7 BV291 SP6  2439
    Ttra dicer1b BV162 SP6  1867
    Ttra tudor1 BV167 SP6  1076
    Ttra argonaute BV168 SP6  323

    Started probe PCR.

    2014-06-05 17.42.43

    09/05/14

    Began probe synthesis at 10:30.

    10/05/14

    Done with probe synthesis, see values above.

     
  • Bruno Vellutini 17:01 on 2014/04/29 Permalink
    Tags: , distal-less, , , , ,   

    Cloning Ttra frizzled, Nano hox1, dlx, wnts 

    Need to clone the remaining frizzleds from Terebratalia and wnts from Novocrania. Set overnight regular PCR with:

    Nano hox1 Nano wnt4 Nano wnt7 Nano dlx Ttra fz5/8 Ttra fz7

    30/04/14

    20140430_175703

    Unfortunately hox1 and the wnts did not work. I should change something in the PCR. I extracted dlx and frizzleds and set a ligation for 4h at RT.

    Transformed and plated the 3 genes and incubated at 37 °C.

    01/05/14

    Pic colonies for colony PCR. Run gel and set bacteria to grow.

    20140501_150448

    02/05/14

    Extracted minipreps and set sequencing PCR.

     
  • Bruno Vellutini 17:20 on 2013/02/22 Permalink
    Tags: distal-less, , , , , ,   

    Terebratalia fluorescent and double in situs for segmentation 

    Starting a new fluorescent and double in situ for segmentation genes. Standard procedures.

    wnt1 wnt5 dlx en (DNP)
    en + wnt1 en + wnt5 en + dlx

    23/02/13

    Probes put at 14h.

    25/02/13

    Washing day. I put the DIG-POD for all wells except en with DNP-POD (HRP).

    26/02/13

    Regular washes.

    27/02/13

    Developed first probe in Cy3 for 1h45. Stopped the reaction 2x 5 min in detergent solution at 62 °C. After washes I checked samples in the stereoscope, but could not see any clear signal in any of the samples. Doubles were blocked and incubated with second antibody (DNP-POD).

    28/02/13

    Developed second probes from doubles in Cy5. Samples were stopped in the same manner and further washed in PTw. I stained with DAPI 1:1000 in PBS for 20 min, washed 3x in PBT and stored in PTw. I mounted 1 slide with en+wnt5 and had a look under the scope.

    It seems that en with DNP-HRP has no background, but the signal was really faint. This might be do re-using the 500 µL of a 0.5 ng/µL probe (find out later that it was half diluted…).

    Snap-5163

    The wnt5 with DIG-POD resulted in a dirty and shiny larva with background all around. Cannot see any real signal. So it seems that DIG-POD needs to be washed longer or there is something wrong with the antibody or TSA kit.

    Snap-5164

    DAPI staining was ok…

    Snap-5165

     
  • Bruno Vellutini 18:32 on 2013/02/15 Permalink
    Tags: distal-less, , , ,   

    Regular and fluorescent in situ for Terebratalia segmentation genes 

    Beginning a regular in situ for the newest wnt7 and wnt11 and for en and dlx to get their patterns. Also testing the DNP-POD for en and DIG-POD for dlx, wnt1, and wnt5. One well will be for a first double in situ with en and wnt5.

    DNP-AP >> en dlx wnt7 wnt11
    DIG-POD >> en (DNP-POD) dlx wnt1 wnt5
    DOUBLE en (DNP-POD) + wnt5 (DIG-POD)

    16/02/13

    All probes diluted to a regular concentration of 1 ng/µL and added at 14h30.

    18/02/13

    Maybe the oven was not rocking? Not sure…

    Did all the washes and added the corresponding antibodies at the following concentrations:

    Anti-Dig-AP 1:5000
    Anti-Dig-POD 1:200
    Anti-DNP-POD 1:500

    19/02/13

    Just washes.

    20/02/13

    Dig-AP started to develop at 12h00. engrailed and distal-less were already well defined in 30 minutes while wnt7 had nothing and wnt11 had a faint band in pedicle lobe. The patterns became strong after a few hours. AP substrate exchanged once and left overnight at 4°C.

    For fluorescent samples… chaos begin. I mistakenly made the TNT buffer with 1% Tween, instead of 0.1%. After developing I split the samples for en, dlx, wnt1, wnt5 into Ptw and detergent treatments. Reactions were stopped with 2x of detergent solution for 2 minutes at 60°C. I also washed the samples with the 1st POD inactivation inadvertently. At the end I see none of the expected expression patterns under the fluorescent lamp. DNP samples seem to have their mesoderm stained while Dig-POD are fluorescent in the gland cells of the epidermis.

    Will incubate the second probe overnight, anyway.

    21/02/13

    Realized I used a regular Cy3 antibody (with Anti-DNP-HRP rabbit) and not the TSA Kit Cy3… for sure this was the issue. I developed the second probe with Cy5, but now I cannot check if it worked, because the red filter is not installed in the scope.

    Ttra_mesoderm

    After developing the second probe for the doubles, the in situ worked! So engrailed-DNP does work well even for the second probe. Pictures taken under the fluorescent scope:

    en_early_larva en_larva

     
  • Bruno Vellutini 12:44 on 2013/02/13 Permalink
    Tags: , distal-less, , , ,   

    Probe synthesis for old and new Terebratalia segmentation genes 

    Transcription reaction set for at 10h30 and precipitation at 17h00:

    ID GENE ENZYME UTP ng/µL
    BV208 en SP6 DIG 135
    BV208 en SP6 DNP 55
    BV209 dlx SP6 DIG 357
    BV211 wnt4 SP6 DIG 351
    BV218 wnt11 SP6 DIG 1234
    BV210 cdx T7 DIG 1450
    BV212 wnt8 T7 DIG 129
    BV213 eve T7 DIG 2269

    14/02/13

    Pellet washing and specs taken, see above. en was the least concentrated one, specially with DNP utp. Will need to re-do it to get more probe.

     
  • Bruno Vellutini 17:17 on 2013/02/07 Permalink
    Tags: , , distal-less, , , , ,   

    Transformation of plasmids for Terebratalia new probes 

    Some of the genes cloned by Yale and Andi will be used for double in situs. We have no probes and there is a limited amount of plasmid solution. So I’m transforming the plasmids again to make more minipreps (and then probes). The genes are:

    Gene ID Kit
    en TTR54 SP6
    dlx Tt-dlx-L2 SP6
    cdx TTR16 T7
    wnt4 TTR164 SP6
    wnt8 TTR104 T7
    eve Tt-eve-L2 T7

    I used 1 µL of plasmids for the transformation and diluted the bacteria solution prior to plating by 1:100 (1µL bacteria: 99µL LB at 37°C). I then plated 50 µL of the 1:100 solution at 16h30.

    08/02/13

    Perfect number of colonies!

    2013-02-08 17.42.06

    09/02/13

    T7
    en BV167
    dlx BV168
    cdx BV169
    wnt4 BV170
    wnt8 BV171
    eve BV172

    10/02/13

    Extracted the plasmids and started the probe PCR.

    11/02/13

    Probe PCR extracted.

    2013-02-11 15.07.35

     
  • Bruno Vellutini 12:52 on 2012/05/10 Permalink
    Tags: distal-less, , , , , , , , ,   

    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.

     
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