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  • Bruno Vellutini 22:57 on 2015/05/11 Permalink
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    Long probe for Novocrania wnt1 


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    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 20:09 on 2015/05/06 Permalink
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    Probe PCR Membranipora final genes 


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    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 18:16 on 2015/03/28 Permalink
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    Probe synthesis Membranipora mesodermal genes 


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    Set probe PCR for the first batch of genes published by the sequencing facility.

    id species gene enzyme ng/µL comment
    BV359 Membranipora membranacea FoxC SP6 1020
    BV361 Membranipora membranacea FoxD SP6 445
    BV362 Membranipora membranacea FoxF SP6  545
    BV363 Membranipora membranacea Mef2 SP6  449
    BV365 Membranipora membranacea Noggin2 SP6  988
    BV367 Membranipora membranacea Eya SP6  826 short isoform
    BV368 Membranipora membranacea Eya SP6  1271 long isoform
    BV370 Membranipora membranacea mPRX SP6  620
    BV372 Membranipora membranacea FHL SP6  321
    BV374 Membranipora membranacea Pax6 SP6  1072
    BV375 Membranipora membranacea Mox T7  1066
    BV376 Membranipora membranacea Wnt4 SP6  1647

    30/03/2015

    Ran gel and extracted probe pcr.

    2015-03-30 17.37.26

    31/03/2015

    Ran and extracted probe pcr for the second batch of sequences.

    2015-03-31 12.19.30

    2015-03-31 12.19.22

    02/04/2015

    Started riboprobe synthesis reaction. Ran for 6.5h and set to precipitate with 5µL of LiCl2.

    03/04/2015

    Precipitate and finish probes. Check above for values.

     
  • Bruno Vellutini 18:15 on 2015/01/27 Permalink
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    Probe synthesis of Terebratalia and Novocrania 


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    Probe synthesis of some longer fragments for Novocrania in situ wnt5, smo, fgf8 and standard Terebratalia clones hh and en.

    SP6 ng/µL
    Na wnt5 BV352  1146.5
    Na smo2 BV354  906.7
    Na fgf8 BV355  964.2
    Tt hh BV129  1510.6
    Tt en TTR54  573.1

    Set probe PCR. Extracted.

    28/01/2015

    Ran gel and extracted.

    2015-01-30 11.05.52

    29/01/2015

    Set probe synthesis and precipitated.

    30/01/2015

    Finished probes.

     
  • Bruno Vellutini 15:29 on 2014/11/25 Permalink
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    Probe synthesis Novocrania delta/notch 


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    Probe PCR set for Novocrania delta (BV348: Sp6) and notch (BV 350: Sp6).

    2014-11-25 16.44.03

    Cut and extracted.

    26/11/14

    Set transcription reaction for 7h. Added DNAse and 5 µL of lithium.

    27/11/14

    Finishing up.

    ng /µL
    Nano delta 1978
    Nano notch 1571
    Tt pou4 371

     

     
  • Bruno Vellutini 19:31 on 2014/11/18 Permalink
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    Probe synthesis of Novocrania Hedgehog pathway 


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    Selected minipreps for probe synthesis of Novocrania Hedgehog pathway and remaining Terebratalia genes. From this post.

    BV326 Terebratalia transversa Lmx1 T7 ok + SP6
    BV328 Terebratalia transversa NK1a T7 ok + SP6
    BV330 Terebratalia transversa NK1b T7 ok + SP6
    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    TTR Terebratalia Pax6 T7 DIG
    TTR Terebratalia Pax6 T7 DNP

    Set probe PCR to run overnight.

    19/11/14

    Ran gel and painfully extracted the PCR…

    2014-11-19 19.09.46

    20/11/14

    Started probe synthesis for the genes above at 9:30. Finished at 16:30 and precipitated overnight.

    21/11/14

    gene ng/µL goal concentration volume total volume volume of hybe buffer
    Ttra lmx1 906 50 24 426 402
    Ttra nk1a 974 50 24 458 434
    Ttra nk1b 1098 50 24 516 492
    Nano ptc1 1075 50 24 505 482
    Nano smo1 2041 50 24 959 936
    Nano smo2 1830 50 24 860 836
    Nano gli 488 50 24 229 206
    Nano pax6 1350 50 24 634 611
    Nano pax37 2224 50 24 1045 1022
    Nano nk1 1050 50 24 494 470
    Ttra pax6 dig 1076 50 24 506 482
    Ttra pax6 dnp 50 50 24 23 0

     

     
  • Bruno Vellutini 10:11 on 2014/07/01 Permalink
    Tags: , probe,   

    Probe synthesis for Membranipora wnts 


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    Setting up a probe PCR for the synthesis of the remaining Membranipora wnt genes’ probes.

    gene id primer ng/µL
    Mmem wnt6 BV312 SP6  575
    Mmem wnt7 BV315 SP6  410
    Mmem wnt8 BV318 SP6  877
    Mmem wnt10a BV321 SP6  963
    Mmem wnt10b BV324 SP6  1089

    10/07/2014

    Done.

     
  • Bruno Vellutini 10:16 on 2014/06/16 Permalink
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    Probe synthesis for Novocrania and Membranipora 


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    Starting probe synthesis (based on) for Novocrania en and pax258 with DNP utp for fluorescent in situ and many Membranipora genes.

    Probe ng/µL
    Nano en DNP 86
    Nano pax258 DNP 59
    Mmem dlx 378
    Mmem gbx 332
    Mmem pax258 1141
    Mmem wnt1 445
    Mmem wnt4b 64
    Mmem wnt5 167

    2014-06-16 18.12.39

    17/06/14

    Run probe synthesis reaction and started probe precipitation.

    18/06/14

    Nice pellets and specs are above.

     
  • Bruno Vellutini 10:11 on 2014/05/08 Permalink
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    Probe synthesis Ttra frizzleds Nano dlx and more 


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    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 11:10 on 2013/11/04 Permalink
    Tags: , probe,   

    Re-synthesis of Novocrania wnt1 and wnt5 probes 


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    Since the probes do not seem to be working for Novocrania in situ I will restart probe synthesis from scratch.

    • Set probe PCR.
    • Run Gel.
    • Extract.

    2013-11-04 17.04.12

    05/11/13

    Transcription reaction for 6h and precipitate at -20 °C overnight.

    06/11/13

    Finished probe synthesis:

    gene ng/µL
    wnt1 3349
    wnt5 185

     
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