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  • Bruno Vellutini 16:49 on 2012/01/09 Permalink
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    MiniPrep for Membranipora PIWIL2 and NANOS 


    Two positive colonies form Mm PIWIL2 (1 and 3) and Mm NANOS (2 and 3) were picked and added to a growth tube with 3 mL of LB medium. Tubes were left in the S8 large shaker overnight at 210 rpm / 37 °C.


    1.5 mL were poured to eppendorfs, centrifuged, discarded supernatant and followed the MiniPrep protocol. DNA was kept in the fridge overnight before the sequencing reaction.


    PCR sequencing reaction and submitted samples:

    ID Gene Primer Quality Probe Kit
    BV1 PIWIL2 T7 bad
    BV2 PIWIL2 T7 bad
    BV3 NANOS T7 ok SP6
    BV4 NANOS T7 ok SP6
    BV5 PIWIL2 SP6 bad
    BV6 PIWIL2 SP6 short, ok SP6


    Sequences came back, see above.

  • Bruno Vellutini 11:37 on 2012/01/09 Permalink
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    New ligation/transformation for Bn PUM1 F2, Mm MAEL F2, Mm DDX4 R2, Mm PIWIL1 R1, Mm PUM, Mm TDRD1 


    Set a new ligation for Bn PUM1 F2, Mm MAEL F2, Mm DDX4 R2, Mm PIWIL1 R1, Mm PUM, Mm TDRD1; + 2 control tubes using the same amount (1.5 µl) of the control DNA insert.


    Transformation using 3 µl of ligation per tube, except for Control 2 where I added 5 µl of ligation. Plated with 400 µl and waited plates to be completely dry to incubate at 37 °C at 19:00.


    Chema suggested to quantify the amount of DNA in the PCR products to be able to calculate the quantity to be used in the ligation reaction. To do this I used NanoDrop spectophotometer.

    Bn PUM1 F2 Mm MAEL F2 Mm DDX4 R2 Mm PIWIL1 R1 Mm PUM Mm TDRD1
    260/280 2.54 2.02 1.87 0.63 2.12 2.21
    260/230 0.77 1.47 0.29 -0.02 0.79 0.41
    ng/µl 56.3 207.7 24.0 -6.2 62.6 64.3

    260/280: ratio of sample absorbance at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.

    260/230: ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. They are commonly in the range of 1.8-2.2. If the ratio is appreciably lower, this may indicate the presence of co-purified contaminants.

    ng/uL: sample concentration in ng/uL based on absorbance at 260 nm and the selected analysis constant.

    From NanoDrop User Guide.

    According to Aina, the contamination at the 260/230 is from the extraction buffer and should not interfere with the ligation.


    Colony growth was better this time, but I still should be getting more colonies than now for some samples. Increase in colony number might be due to the plating amount (400 µl) or even the 1 µL increase in the PCR product for the ligation.

    Gene Colonies Picked
    Bn PUM1 F2 ~10 6
    Mm MAEL F2 3 3
    Mm DDX4 R2 Many 6
    Mm Piwil1 R1 5 5
    Mm PUM ~15 6
    Mm TDRD1 ~10 6
    Control 1 7 5
    Control 2 Many 5

    Colony checking PCR started at 14:00.


    No positive colonies… Repeat.

    Ms_Colony2_110112MmColony 2012-01-11 12hr 23min

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