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  • Bruno Vellutini 20:00 on 2013/04/30 Permalink
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    Priapulus RNA extraction of specific stages 

    Samples are neutrally buoyant with RNAlater, which makes picking a pain. I added 1x PBS with DEPC water to make the embryos sink, but still had to spin them at least 3 times at 8rpm to get all of them. Also, they stick along the wall making it hard for pipeting. In addition I spin the RNAse free tube with samples to dry out until maximum of 100µL, less when possible. Afterwards the protocol went ok.

    However, after centrifuging the isopropanol added sample a rock solid cement was fixed in the bottom of the tube. Pellet like flakes were visible with the ethanol wash and I saw them dissolving with the addition of DEPC water.

    These were the NanoDrop measurements:

    Sample ID ng/ul 260/280 260/230
    Pc oo 152,94 1,65 0,47
    Pc 1d 106,72 1,49 0,48
    Pc 2d 306,38 1,71 0,63
    Pc 3d 264,63 1,64 0,58
    Pc 4d 218,24 1,71 0,53
    Pc 5d 225,42 1,64 0,55
    Pc 6d 167,43 1,57 0,51
    Pc 7d 81,00 1,55 0,58
    Pc 9d 249,42 1,56 0,58
     
  • Bruno Vellutini 19:00 on 2013/04/30 Permalink
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    Cystein treatment for 12/04/13 (18d) and 24/04/13 (6d) egg masses. When opening the egg mass for 18d many juveniles poped-out  and thus received the cystein treatment directly.

    01/05/13

    Many juveniles (18d) burst because of the cystein treatment. I will need to fix this stage again. Otherwise, the earlier 6d embryos look fine.

     
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