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  • Bruno Vellutini 18:00 on 2012/04/30 Permalink
    Tags: debug,   

    Debugging contamination of Wnts 


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    Four of the sequences from 2012-04-25_0446 were contamination of Terebratalia Wnts in my samples. Wnts had already appeared in the sequences of Henrike, but the source was not identified. To identify if the contamination is in the sequencing reaction or the actual Wnts are being cloned Chema suggested to run a PCR with the miniprep using the gene specific primers. I also included the specific Terebratalia primers to see if there is any amplification.

    ID Gene Actual sequence
    BV93 Mm Piwi1 Tt Wnt16
    BV95 Mm Piwi1 Tt Wnt10
    BV99 Tt Nanos Tt Wnt10
    BV104 Tt Nanos Tt Wnt16

    Debug PCR for Wnt contamination

    The PCR shows that the actual Wnts were being cloned. LB medium used for transforming or growing should be the culprit.

     
  • Bruno Vellutini 18:00 on 2012/04/30 Permalink
    Tags: , , , , , ,   

    Probe synthesis for Mm Vasa, Nanos, Piwi1, Piwi2 and Tt Vasa, Nanos, Piwib 


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    Running a probe PCR for germ line genes of Membranipora (again) and Terebratalia (see table below). Setup is the same as a colony PCR, but doubling the amount (50µL) and using 3 µL of plasmid DNA. I extracted the bands and purified the DNA.

    Membranipora probe pcr for nanos, piwi2, vasa, and piwi1Terebratalia probe pcr for vasa, nanos, and piwib

    01/05/12

    This is the gel with the purified yield and the nanodrop measures.
    Purified dna from probe pcr of Mm and Tt germ genes

    02/05/12

    Transcription reaction initiated at 11h, tubes put in the cycler using incubate function at 37 °C.

    03/05/12

    Finished the probe synthesis and stored in hybe buffer diluted 50 ng/µL.

    Probe specs
    ID Gene Probe Kit Probe PCR ng/µL  Probe ng/µL
    BV3 MmNanos SP6 881.0 1283.41
    BV6 MmPiwi2 SP6 745.1 1149.77
    BV91 MmVasa SP6 586.9 477.97
    BV94 MmPiwi1 SP6 571.3 902.3
    BV96 TtVasa SP6 517.8 572.53
    BV100 TtNanos SP6 582.4 1158.87
    BV103 TtPiwib SP6 651.8 493.36

     
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