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  • Bruno Vellutini 20:09 on 2015/05/06 Permalink
    Tags: , , , pcr, ,   

    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 20:07 on 2015/05/06 Permalink
    Tags: , , pcr,   

    Novocrania wnt1 one more time 


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    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

    2015-05-07 11.35.14

    It worked. Extracted, ligated, transformed and plated.

    08/05/15

    Colony PCR and 2 volonies were fine:

    2015-05-08 17.18.24

    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 13:36 on 2015/01/19 Permalink
    Tags: , , , , pcr, ,   

    Cloning more segmentation genes 


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    I selected alternate primers for getting longer fragments and improving the quality of Novocrania in situs.

    Nano ptc2, wnt1, smo1, smo2, fgf8, Mmem wnt4

    2015-01-20 11.02.32

    wnt1 did not work again, bad ptc2 again, smo1 and smo2 weakly worked, fgf8 worked, Mmem wnt4 not… Re-run Nano ptc2 with different primers:

    2015-01-20 17.51.33

    Combination of primers 3F and 2R (395 bp fragment) was the only one that worked. Transformed:

    2015-01-23 17.16.01

    T7
    Na wnt5 BV352
    Na wnt5 BV353
    Na smo2 BV354
    Na fgf8 BV355
    Na fgf8 BV356
    Na ptc2 BV357
    Na ptc2 BV358

     

     
  • Bruno Vellutini 15:31 on 2014/11/25 Permalink
    Tags: , , pcr,   

    Cloning Novocrania patched and longer clones of wnt1, wnt5 


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    I made new primers for ptc2 and longer clones for wnt1 and wnt5. Set PCR overnight.

    26/11/14

    Ran gel and cut a band, but wnt1 and ptc2 did not work… OF COURSE! because fragment lengths are the following: wnt1=1239 bp, wnt5=1097 bp, ptc2=1258 bp! And the elongation time of the PCR was set to X.

    2014-11-26 11.16.21

    28/11/14

    Re-tried the same PCR with a longer elongation time. The result was the same. Chema suggested running nested primers to see if something appears.

    2014-11-28 17.18.15

     
  • Bruno Vellutini 18:33 on 2014/11/07 Permalink
    Tags: , , , , , , , pcr   

    Novocrania cloning 


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    Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.

    08/11/14

    Ran gel and set ligation.

    2014-11-08 15.29.44

     
  • Bruno Vellutini 17:31 on 2014/09/09 Permalink
    Tags: , , , , pcr,   

    Round of Terebratalia cloning 


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    I have ordered additional primers to clone more segmentation genes. Setting a PCR with 25 µM for primer concentration and 65 °C for annealing temperature.

    Ttra lmx1, nk1a, nk1b, odd

    10/09/14

    2014-09-10 12.10.25

    So, odd failed.

    Set ligation at 4 °C to go overnight (have to write…)

     
  • Bruno Vellutini 18:36 on 2014/06/06 Permalink
    Tags: , , pcr   

    Cloning Membranipora Wnts 


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    Set PCR.

    07/06/14

    Gel, extraction, ligation, transform.

    09/06/14

    Set colony PCR overnight.

    10/06/14

    Ran gel and put colonies to grow.
    2014-06-10 12.25.20

    11/06/14

    Extracted minipreps and did sequencing PCR. Left samples at the sequencing facility.

     
  • Bruno Vellutini 01:01 on 2013/09/10 Permalink
    Tags: , , pcr,   

    Novocrania one more time 


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    Using Andi’s primers with annealing temperature at 57 °C.

    11/09/13

    Engrailed rendered a band at the expected size (638 bp), although it is a bit spread band. It is quite possible that wnt1 had a band at the right place (718 bp), but it was very weak; I cut it anyway. Wnt5 band was also at the correct place (932 bp), but also weak.

    2013-09-11 16.37.23

    Set ligation reaction at 16h and stored at 4°C.

    12/09/13

    Transformation and plating.

    13/09/13

    en had a lot of colonies, wnt1 had a normal amount, and wnt5 had a few colonies. I managed to do 10 colonies for the first two and 8 colonies for wnt5:

    2013-09-16 10.57.37

    16/09/13

    Picked colonies for minipreps

    gene colony T7
    en 8 BV266
    en 9 BV267
    en 10 BV268
    wnt1 1 BV269
    wnt1 2 BV270
    wnt1 3 BV271
    wnt5 1 BV272
    wnt5 2 BV273
    wnt5 3 BV274

    17/09/13

    • Extracted plamids.

    18/09/13

    • Set sequencing pcr at 10:30.
    • Dropped sequences at the facility at 14:30.

    22/09/13

    Checked the alignment and it looks good! See files: Nano_en-wnt1-wnt5

     
  • Bruno Vellutini 18:04 on 2013/08/28 Permalink
    Tags: , , pcr,   

    Novocrania en, wnt1, wnt5 


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    Set PCR with 50 °C annealing temperature and 40 cycles with new primers.

    29/08/13

    2013-08-29 11.55.15

    Not good enough, I cut the top band of wnt1 anyway…

     
  • Bruno Vellutini 18:08 on 2013/08/14 Permalink
    Tags: , , pcr,   

    Novocrania cloning en, wnt1, wnt5 


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    Did a new PCR with new primers for engrailed, wnt1, and wnt5 in Novocrania at 55 °C.

    15/08/13

    It failed…

    2013-08-15 17.39.43

     
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