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  • Bruno Vellutini 18:45 on 2015/03/30 Permalink
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    Membranipora wnt in situ 

    First new try with bryozoan in situ. Separate wells for early (until extended gastrula) and late stages (early cyphonautes and later cyphonautes).

    early stages 1x nk2.1 1x wnt1 1x wnt5 1x wnt7 1x wnt8
    late stages 0.1x nk2.1 0.1x wnt1 0.1x wnt5 0.1x wnt7 0.1x wnt8
    early stages 1x dlx 1x gbx 1x pax258 1x nanos 1x vasa
    late stages 0.1x dlx 0.1x gbx 0.1x pax258 0.1x nanos 0.1x vasa

    After protk step (10 min) embryos were suspended in glycine (added 800 µL) and did not sink. I added 600 µL of glycine for the second wash. The whole glycine steps lasted for 15 min. The remaining washes were a bit longer than 5 min, but I could control it better.

    I noticed that the embryonic membrane dissolves with protk.

    Let overnight in pre hybe.

    31/03/2015

    Diluted and added probes to in situ (first 0.1x then 1x). Left oven set to 63 °C (62 in the electronic thermometer and 63 in the mercury thermometer).

    02/04/2015

    First hybe wash I removed 1x probes first, filled with warm hybe wash and then removed 0.1x probes and added warm hybe.

    • 1x 10min hybe wash
    • 1x 50min hybe wash
    • 1x 30min 75% hybe
    • 1x 30min 50% hybe
    • 1x 30min 25% hybe
    • 1x 30min 2x SSC
    • 3x20min 0.2x SSC
    • 1x 10min 75% 0.2x SSC
    • 1x 10min 50% 0.2x SSC
    • 1x 10min 25% 0.2x SSC
    • 1x 10min PTw.
    • 4×10-15min washes in PBT (total 45min)
    • 1h in 1x blocking buffer
    • Overnight at 4°C with anti-dig-ap

    03/04/2015

    • 3x 5min PBT
    • 4x 15min PBT
    • 5x 30min PTw

    Developed in situ. A significant amount of pinkish background came up in my probes. wnt5 was fast, also dlx. Others had different levels of background in addition to the real signal, except Chema’s probe for NK2.1. Thus, this background is likely due to probe design or probe synthesis. Here are the lengths of the clones used to synthesize the probes:

    gene nk2.1 wnt1 wnt5 wnt7 wnt8 dlx gbx pax2/5/8 nanos vasa
    bp 1113 1038 1073 869 886 823 888 902 868 812

    Length of the probe can be the issue but it does not quite explain the rapid background coming up. Longer probes should result in a better signal to noise ratio because the signal comes up faster and not because the background comes up slower. It can also be something related to the probe synthesis process. But what? I usually get high yields and good peak ratios. Check the numbers to see!

    Ah, one obvious thing is the probe concentration. NK2.1 probe is probably more diluted than the others since it is its second or third use. Thus, the next in situ should be lower concentration or higher temperature.

    04/04/15

    Continued developing wnt7 and gbx and stopped them after 3h. Ethanol washes with 20min in 100% and let in PTw overnight at 4° C because glycerol was not ready.

    05/04/15

    Putting them in 70% glycerol with DAPI. Will put apart some of the NK2.1 embryos to do a MAPK immuno staining.

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

    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 18:39 on 2015/03/23 Permalink
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    Cloning Membranipora mesodermal genes 

    Cloning mesodermal genes in Membranipora: wnt4, pax6, noggin, mox, paraxis, foxf, mprx, nog2, mef2, fhl, foxc, foxd, eya. Nano wnt1 longer probe 2try.

    24/03/15

    2015-03-23 19.57.29

    25/03/15

    2015-03-25 16.45.11

    26/03/15

    Extract minipreps and sequencing. Done. BV359 to BV377 with T7 primer.

    BV359 Membranipora membranacea FoxC T7 ok + SP6
    BV360 Membranipora membranacea FoxC T7 ok + SP6
    BV361 Membranipora membranacea FoxD T7 ok + SP6
    BV362 Membranipora membranacea FoxF T7 ok + SP6
    BV363 Membranipora membranacea Mef2 T7 ok + SP6
    BV364 Membranipora membranacea Mef2 T7 ok + SP6
    BV365 Membranipora membranacea Noggin2 T7 ok + SP6
    BV366 Membranipora membranacea Noggin2 T7 ok T7
    BV367 Membranipora membranacea Eya T7 ok + SP6 short isoform
    BV368 Membranipora membranacea Eya T7 ok + SP6 long isoform
    BV369 Membranipora membranacea Eya T7 ok T7
    BV370 Membranipora membranacea mPRX T7 ok + SP6
    BV371 Membranipora membranacea mPRX T7 ok T7
    BV372 Membranipora membranacea FHL T7 ok + SP6
    BV373 Membranipora membranacea Pax6 T7 ok T7
    BV374 Membranipora membranacea Pax6 T7 ok + SP6
    BV375 Membranipora membranacea Mox T7 ok T7
    BV376 Membranipora membranacea Wnt4 T7 ok + SP6
    BV377 Membranipora membranacea Wnt4 T7 ok + SP6

     
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