phd.brunovellutini.comPhD notebook by Bruno C. Vellutini

PhD notebook by Bruno C. Vellutini Search for: About Hello. This is the electronic laboratory notebook of my PhD. I used WordPress with the P2 theme to take notes and document experiments. Overall, it worked really well. At the end of my PhD, I printed all posts for local storage and the digital version remains online. I come back here sometimes to check how I did things. Thanks for passing by. You can find me on brunovellutini.com . Recent Posts Membranipora simple staining Membranipora in situ + mapk, a re-try Membranipora MAPK staining on in situ embryos Novocrania wnt1 in situ with longer probe Long probe for Novocrania wnt1 Membranipora in situ and Novocrania wnt1 Probe PCR Membranipora final genes Novocrania wnt1 one more time Membranipora cloning final genes Membranipora MAPK immuno staining Archives August 2015 (3) May 2015 (5) April 2015 (3) March 2015 (3) February 2015 (4) January 2015 (3) November 2014 (11) October 2014 (1) September 2014 (11) July 2014 (4) June 2014 (26) May 2014 (4) April 2014 (22) March 2014 (4) January 2014 (7) December 2013 (3) November 2013 (6) October 2013 (8) September 2013 (18) August 2013 (31) July 2013 (4) June 2013 (4) May 2013 (10) April 2013 (16) March 2013 (17) February 2013 (10) November 2012 (10) October 2012 (7) September 2012 (6) August 2012 (13) July 2012 (8) June 2012 (5) May 2012 (23) April 2012 (20) March 2012 (9) February 2012 (5) January 2012 (13) December 2011 (5) November 2011 (5) July 2011 (5) February 2011 (1) Calendar May 2024 M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 « Aug Categories entry (373) Recent tags Membranipora membranacea ( 140 ) confocal ( 23 ) mapk ( 26 ) even-skipped ( 4 ) foxa ( 5 ) nk2.1 ( 1 ) six3/6 ( 6 ) gata ( 1 ) pl10 ( 6 ) probe ( 24 ) Novocrania anomala ( 43 ) wnt1 ( 3 ) Terebratalia transversa ( 79 ) runx ( 7 ) in situ ( 43 ) distal-less ( 11 ) nanos ( 25 ) fgf ( 2 ) piwi ( 32 ) chordin ( 3 ) vasa ( 22 ) bmp ( 3 ) pcr ( 37 ) wnt ( 60 ) pax6 ( 3 ) cloning ( 15 ) fgf8/17/18 ( 4 ) staining ( 5 ) foxc ( 2 ) fhl ( 2 ) noggin ( 2 ) eya ( 2 ) mprx ( 2 ) foxd ( 2 ) mox ( 2 ) Recent comments BLASTer script updat… on First dirty script Meta Log in Entries feed Comments feed WordPress.org Recent Updates Toggle Comment Threads | Keyboard Shortcuts Bruno Vellutini 16:37 on 2015/08/25 Permalink Tags: confocal ( 23 ) , Membranipora membranacea ( 140 ) Membranipora simple staining Just a simple staining of mixed stages of Membranipora using DAPI and Phalloidin 647 for morphology. 3x 30min 0.2% PTx washes. 1x PTx overnight at 4°C. 26/08/15 Staining with Phalloidin and DAPI for 2h. Let embryos in PBS+DAPI 1:1000 at 4°C. It failed… weird. Bruno Vellutini 12:09 on 2015/08/25 Permalink Tags: mapk ( 26 ) , Membranipora membranacea ( 140 ) Membranipora in situ + mapk, a re-try Since I saved some de-glycerolled embryos I’ll repeat the immuno staining with a longer developing time. gata b nk2.1 Washed 1h in PBT (4x). Washed 3h in 5% NGS (3x). Added anti-mapk antibody 1:100. Let overnight at 4°C. 26/08/15 Washed primary antibody with 3x PBT 5min + 3x 30min. 5% NGS 3x 30 min. Added both anti-mouse-pod/hrp at 1:250 for 4h at room temp. Washed 4x 5min with PBT and let overnight at 4°C. 27/08/15 Wash 1x PBT. Wash 3x TNT buffer. Develop with cy3 for 5min. Wash with PBS (no detergent solution). Embed in TDE. Bruno Vellutini 11:47 on 2015/08/18 Permalink Tags: confocal ( 23 ) , even-skipped ( 4 ) , foxa ( 5 ) , gata, mapk ( 26 ) , Membranipora membranacea ( 140 ) , nk2.1, six3/6 ( 6 ) Membranipora MAPK staining on in situ embryos Selected 5 genes for a MAPK immuno staining to resolve the spatial relationship between MAPK vegetal blastomere, gene expression and the embryonic axes. gata_b foxa six3/6 evx nk2.1 I started to de-glycerol embryos in hourly washes of PTw around 1200. 20/08/2015 3x 10min PTx 2x 45 PBT 2x 30min PTx+NGS 1:200 anti-mapk antibody added at 14:00 21/08/2015 Wash primary antibody (afternoon) 3x 5min. 4x 30min. Add secondary antibody anti-mouse pod at 1:250 dilution at 1800. 22/08/2015 3x 5min PBT washes. 4x 30min PBT washes. Overnight at 4°C in PBT. 23/08/2015 Washed 3x 5min in TNT buffer. Developed in 50 µL TSA + Cy5 fluorochrome for 3 min. Washed 10min in detergent solution at 67°C and another with warm detergent but at room temp to cool down. Washed 2x 5min PBS. TDE series ending with 97% TDE in PBS with DAPI 1:1000. 24/08/2015 Mounted slides and checked under scope. There is quite some background with the orange filter for cy3. I cannot distinguish any signal at the posterior vegetal blastomere. Confocal showed that there was no signal from the MAPK antibody. Maybe the longer time in glycerol messed the epitopes? Or the time developing (3min) was not enough… DAPI (cyan), MAPK (magenta), in situ (yellow): gata b gata b gata b gata b nk2.1 nk2.1 nk2.1 Bruno Vellutini 11:13 on 2015/05/12 Permalink Tags: in situ ( 43 ) , Novocrania anomala ( 43 ) , runx ( 7 ) , Terebratalia transversa ( 79 ) , wnt1 ( 3 ) Novocrania wnt1 in situ with longer probe Na wnt1 Tt runx Did everything as usual and embryos looked normal. 13/05/15 Embryos quite damaged, including Terebratalia. Possible that pipette is not well calibrated? Restarted in situ today with extreme gentleness. Added 5 µL protk in 10 mL (instead of 2.5 in 5 µL) using the P20 pipette. Fosfatase wash with 83 °C and added hybe buffer around 1800. 14/05/15 Add probe. Bruno Vellutini 22:57 on 2015/05/11 Permalink Tags: Membranipora membranacea ( 140 ) , Novocrania anomala ( 43 ) , pl10 ( 6 ) , probe ( 24 ) , wnt1 ( 3 ) Long probe for Novocrania wnt1 Set probe PCR for: Na wnt1 BV387: T7 80 ng/µL Mm pl10 BV79: SP6 702 ng/µL and extracted. 12/05/15 Set transcription reaction and precipitation. 13/05/15 Finished probes (see values above) and made 1x dilution for wnt1. Bruno Vellutini 18:24 on 2015/05/08 Permalink Tags: bmp ( 3 ) , chordin ( 3 ) , distal-less ( 11 ) , fgf ( 2 ) , in situ ( 43 ) , Membranipora membranacea ( 140 ) , nanos ( 25 ) , Novocrania anomala ( 43 ) , piwi ( 32 ) , pl10 ( 6 ) , vasa ( 22 ) , wnt1 ( 3 ) 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: bmp ( 3 ) , chordin ( 3 ) , fgf ( 2 ) , pcr ( 37 ) , piwi ( 32 ) , probe ( 24 ) Probe PCR Membranipora final genes Probe PCR for bmp/24, chordin, fgf8 and piwi1. 07/05/15 Extracted probe PCR gel. 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 20:07 on 2015/05/06 Permalink Tags: Novocrania anomala ( 43 ) , pax6 ( 3 ) , pcr ( 37 ) , wnt ( 60 ) Novocrania wnt1 one more time Set PCR with 60°C annealing, 1:45 elongation, 40 cycles and primers at 25mM. wnt1 1:10 cDNA wnt1 1:20 cDNA pax6 1:10 cDNA pax6 1:20 cDNA 07/05/15 It worked. Extracted, ligated, transformed and plated. 08/05/15 Colony PCR and 2 volonies were fine: Put these to grow under the IDs: T7 Na wnt1 long BV386 Na wnt1 long BV387 09/05/15 Extract miniprep and set sequencing reaction. Bruno Vellutini 18:29 on 2015/04/29 Permalink Tags: bmp ( 3 ) , chordin ( 3 ) , cloning ( 15 ) , fgf8/17/18 ( 4 ) , Membranipora membranacea ( 140 ) , nanos ( 25 ) , piwi ( 32 ) , vasa (...