Updates from January, 2012 Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 21:59 on 2012/01/11 Permalink
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    Debugging PCRs with Bn PUM1 F2, Mm MAEL F2, Mm PUM, Mm TDRD1 

    11/01/12

    RACE PCR: Bn PUM1 F2, Mm MAEL F2

    PCR: Mm PUM, Mm TDRD1

    BnMmPCRs 2012-01-11 19hr 04min

    I cut the bands from the RACE PCR very quickly under UV light (<10s each) and purified regular PCR directly (band was not cut from the gel). This is to be sure that exposure to UV is not damaging the samples, which could be responsible for the ligation failures.

    Standard ligation set up at 21:00 (protocol values).

    12/01/12

    Heat shock transformation and plating at 17:30, looking good.

    13/01/12

    Bn PUM1 F2 and Mm MAEL F2 did not have many colonies (6 and 3, respectively). The latter could be because of the amount of PCR product interfering in the ligation. Mm PUM and Mm TDRD1 had extra colonies and I included 6 of each in the colony check PCR, started at 10:50.

    MmColony 2012-01-13 15hr 17min

    Positive colonies! Looks like purifying PCR product directly is more worth it, if there is only one band. Apparently the UV was indeed damaging the DNA from the samples. Asterisks marks are colonies to be picked for the MiniPreps.

    Bn PUM1 F2 No positive colonies, only a 500 bp one.
    Mm MAEL F2 Positive: 1, 2, 3
    Mm PUM Positive: 3, 4, 6
    Mm TUDOR Positive: 3, 4, 5 (2 also, but bit shorter)

    15/01/12

    Add 3 mL of LB to MiniPrep tubes and leave in the shaker.

     
  • Bruno Vellutini 18:00 on 2012/01/11 Permalink
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    Extra heat shock transformation 

    11/01/12

    I did an additional heat shock transformation using new Aina’s newest cells and the most recent ligation. Used the following samples Bn PUM1 F2, Mm MAEL F2, Mm PUM, Mm TDRD1, Control 1 (1.5 ┬Ál of ligation mix, what was left) and plated at 17:00.

    12/01/12

    Not enough colonies.

     
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