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  • Bruno Vellutini 11:13 on 2012/01/05 Permalink
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    Membranipora PCR repetition 

    Since the ligation was not working well I am repeating the PCR for PIWIL2, NANOS, PUM1, and TRDR1 — previously done here using the same settings. The only difference is that I set the elongation time to 2 min and not 4 min. Also, I will not add extra loading dye for running the gel, as I mistakenly did last time.


    PCR started 11:00 and the bands looked ok in the gel. Mostly identical to the previous one:

    Membranipora PCR for Piwi, Nanos, Pumilio, and Tudor

    I extracted the samples and set up a ligation starting at 20:00 overnight at 4 °C.


    Heat shock transformation and plating at 18:30.


    All plates had colonies as shown below:

    Gene Colonies
    PIWIL2 16
    NANOS 10
    PUM 5
    TDRD1 3

    PCR for colony testing started at 14:20.

  • Bruno Vellutini 18:38 on 2012/01/04 Permalink
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    RACE PCR for Membranipora and Bugula 


    Repeating RACE PCR for Bugula genes tried previously since only Bn PUM1 F2 was amplified properly according to the PCR product check. Genes from Membranipora will also be done. Genes and primers (7 reactions):

    Bugula Membranipora
    Bn PUM1 F1 Mm PIWIL1 R1
    Bn PUM1 F2 Mm PIWIL1 R2
    Bn PUM1 R1 Mm DDX4 R1
    Bn PUM1 R2 Mm DDX4 R2
    Bn PIWIL1 R1 Mm MAEL F1
    Bn PIWIL1 R2 Mm MAEL F2
    Mm MAEL R1
    Mm MAEL R2

    1st round started at 13:00; 2nd round started at 17:40.


    Agarose gel for Bugula neritina:

    Bugula RACE Pumilio and Piwi

    Only Bn PUM1 F2 had a strong band, the gel was quite similar to the previous RACE PCR run. The faint band marked with “…” Mn PUM1 R2 was cut and frozen if needed in the future.

    Gel for Membranipora membranacea:

    Membranipora RACE Maelstron, Vasa, Piwi

    Mn MAEL F2 had a strong band, as well as Mm DDX4 R2 and Mm PIWIL1 R1. I cut and frozen the short and faint bands Mm MAEL R2 and Mm PIWIL1 R2.

    Extraction and ligation done for Bn PUM1 F2, Mm MAEL F2, Mm DDX4 R2, and Mm PIWIL1 R1 at 17:00 and left at 4 °C overnight. Extra pipetting for mixing ligation components.


    Heat shock transformation and incubated the 4 plates at 37 °C.


    No colonies grew on the plates :( Actually one colony for Bn PUM1 F2. Kevin suggested to do the plating quickly and to not dry out the cells.

    I repeated the transformation using the ligation from yesterday, while transforming the regular PCR from Membranipora. Plating at 18:30.


    Again the number of colonies was low, only one colony for Bn PUM1 F2 and Mm DDX4 R2. Ligation and transformation needs to be repeated. Started a colony testing for both.


    Repeat the ligation for these 4 samples using the ligation control.

  • Bruno Vellutini 17:59 on 2012/01/04 Permalink
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    Colony testing for PUM and PIWI 

    Aina repeated the ligation for Nanos, Pumilio, and Piwi from the regular PCR of Membranipora. No colonies grew for Nanos. From the few colonies in the other two plates only one colony had the plasmid with the gene: Piwi 7.

    Membranipora colony testing

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