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  • Bruno Vellutini 18:29 on 2015/04/29 Permalink
    Tags: , , cloning, , , , ,   

    Membranipora cloning final genes 


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    Set PCR with:

    Mm bmp2/4 Mm otx1 Mm chordin Mm fgf8/17/18
    Mm nanos Mm piwi1 Mm piwi2 Mm vasa

    30/04/15

    Ran gel:

    2015-05-01 16.23.27

    Most important genes worked: bmp2/4, chordin and fgf8. piwi1 is bonus. Extract these and set a ligation for 3h. Transformed and plated around 18h.

    01/05/15

    Set colony PCR.

    2015-05-01 16.23.36

    Put colonies to grow.

    02/05/15

    Extracted minipreps, set sequencing PCR and dropped at seq facility.

    BV378 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV379 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV380 Membranipora membranacea Chordin T7 ok + SP6
    BV381 Membranipora membranacea Chordin T7 ok + SP6
    BV382 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV383 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV384 Membranipora membranacea Piwi1 T7 ok + SP6
    BV385 Membranipora membranacea Piwi1 T7 ok + SP6

     

     

  • Bruno Vellutini 13:36 on 2015/01/19 Permalink
    Tags: cloning, , , , , ,   

    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: cloning, , ,   

    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 19:26 on 2014/11/18 Permalink
    Tags: cloning, , , , , , ovo, , ,   

    Transformation for remaining Terebratalia and Novocrania 


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    Terebratalia: ovo, serrate, pygopus, legless (from here)

    Novocrania: notch, delta, gbx (from here)

    Transformed and plated with 400 µL.

    19/11/14

    Colony PCR.

    2014-11-19 19.09.53

    Set Nano delta and notch to grow.

    20/11/14

    Extracted minipreps for Nano notch and delta:

    gene id seq primer antisense
    Nano delta BV348 T7 SP6
    Nano delta BV349 T7 SP6
    Nano notch BV350 T7 SP6
    Nano notch BV351 T7 T7

    Set sequencing PCR for them.

    24/11/14

    Collected sequences. See orientation above.

     

  • Bruno Vellutini 16:15 on 2014/11/10 Permalink
    Tags: cloning, , ,   

    Cloning Hedgehog components of Novocrania 


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    PCR with diluted primers for 40 cycles instead of 34 for: ptc1, ptc2, smo1, smo2, gli

    2014-11-10 22.32.27

    Had two bands for some. Set a ligation overnight for all at 4 °C.

     

  • Bruno Vellutini 19:19 on 2014/11/06 Permalink
    Tags: cloning, , ,   

    Testing new Novocrania cDNA 


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    I’m testing the new cDNA for Novocrania made by Daniel. He made a mix of extractions from different stages and diluted 1:10 and 1:20. I tested with previous genes that worked, pax2/5/8 and wnt11. Set the PCR with a reaction for the 1x, 1:10 and 1:20.

    07/11/2014

    2014-11-07 12.17.42

     

  • Bruno Vellutini 18:02 on 2014/11/06 Permalink
    Tags: cloning, , , opa, , rhomboid, ,   

    Cloning additional segment polarity in Terebratalia 


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    Cloning of further Terebratalia segment polarity genes: opa, ovo, rho, serrate and genes I saw at the EuroEvoDevo: legless, pygopus.

    07/11/2014

    2014-11-07 12.17.29

     

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

    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 18:43 on 2014/07/01 Permalink
    Tags: cloning, , , ,   

    Cloning genes that failed 


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    Mmem wnt4a Nano hox1 Nano wnt4 Nano wnt7

    PIX

    Well, not that different… Membranipora gene worked somehow, a weak band.

    Set a ligation for Mmem wnt4a and Na hox1.

    03/07/14

    Heat shock transformation and plating.

     

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

    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.

     

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