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  • Bruno Vellutini 16:45 on 2012/05/10 Permalink

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    Fixation of 7 d old L. ruber for confocal and in situ. Bissected the halves of egg masses from previous fixation and removed the outer case. Instead of treating with cystein I simply used the large plastic pipet to dissociate the eggs from the jelly. It worked quite well, even though the jelly would stick in the pipet.

    Eggs were release from the bundle with needles and fixed with the standard protocol for in situ and confocal.

  • Bruno Vellutini 12:52 on 2012/05/10 Permalink
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    Cloning Mm six3/6 and Tt gli, en, dlx, nk6, smo, piwia, wnt7 

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    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.


    In the end the re-PCR did not worked well, so I purified the DNA directly forĀ  Mm six3/6 and Tt piwia, nk6, and smoothened. Ligation was set for these and let over the weekend.


    Heatshock transformation and plating at 18h.


    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


    Set a sequencing PCR at 11:40; delivered at 19:45.


    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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