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  • Bruno Vellutini 08:00 on 2012/01/31 Permalink
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    8th MIC confocal microscopy course at the Dept. of Biomedicine, University of Bergen 


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    Confocal course: 31st of January – 3rd of February 2012. Program: Program and info 8th confocal course 2012

     
  • Bruno Vellutini 20:51 on 2012/01/20 Permalink
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    Membranipora NANOS and PIWIL2 in situ 


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    20/01/12

    Day 1…

     
  • Bruno Vellutini 20:17 on 2012/01/19 Permalink
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    Probe PCR for Membranipora NANOS, PIWIL2, MAEL F2, PUM, TDRD1 


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    Clones amplified for riboprobe synthesis:

    Gene ID Probe Kit
    NANOS BV3 SP6
    PIWIL2 BV6 SP6
    MAEL BV9 SP6
    PUM BV21 T7
    TDRD1 BV23 T7

    Probe PCR: 12:00. DNA extraction and purification: 18:30.

    20/01/12

    Agarose gel was ok, products had the expected length.

    Probe Mm 2012-01-20 10hr 20min

    Quantification of PCR product was also ok:

    Sample ng/µl 260/280 260/230
    NANOS 574.73 1.91 1.92
    PIWIL2 480.20 1.94 1.35
    MAEL 300.42 2.05 0.56
    PUM 293.22 2.00 0.53
    TDRD1 224.36 2.00 0.48

    Mm ProbePCR 20.01.12

    Transcription started at 13:30.

     
  • Bruno Vellutini 16:35 on 2012/01/15 Permalink
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    MiniPrep for Membranipora MAEL F2, PUM, TUDOR 


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    Picked positive colonies to grow. 9 samples on shake to grow at 16:30.

    16/01/12

    Sequencing reaction at 12:00 and tubes dropped by the sequencing facility at 15:00.

    ID Gene Vector Quality Probe Kit
    BV7 MAEL F2 T7  bad  –
    BV8 MAEL F2 T7  ok  SP6
    BV9 MAEL F2 T7  ok  SP6
    BV10 PUM T7  ok  SP6
    BV11 PUM T7  ok  T7
    BV12 PUM T7  ok  T7
    BV13 TUDOR T7  ok  T7
    BV14 TUDOR T7  ok  T7
    BV15 TUDOR T7  ok  T7
    BV16 MAEL F2 SP6  bad  –
    BV17 MAEL F2 SP6  ok  SP6
    BV18 MAEL F2 SP6  ok  SP6
    BV19 PUM SP6  ok  SP6
    BV20 PUM SP6  ok  T7
    BV21 PUM SP6  ok  T7
    BV22 TUDOR SP6  ok  T7
    BV23 TUDOR SP6  ok  T7
    BV24 TUDOR SP6  ok  T7

    19/01/12

    Sequences came back.

     
  • 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 


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    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 


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    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 µl of ligation mix, what was left) and plated at 17:00.

    12/01/12

    Not enough colonies.

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


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    09/01/12

    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.

    10/01/12

    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.

    11/01/12

    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

    13/01/12

    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 


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    08/01/12

    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.

    09/01/12

    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.

    NanoDrop

    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.

    10/01/12

    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.

    11/01/12

    No positive colonies… Repeat.

    Ms_Colony2_110112MmColony 2012-01-11 12hr 23min

     
  • Bruno Vellutini 15:02 on 2012/01/07 Permalink
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    Colony testing for Mm PIWIL2, NANOS, PUM, TDRD1, and Bn PUM1, DDX4 


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    Colony picking and testing for the following genes from the RACE PCR and regular PCR with Membranipora genes. PCR started at 14:20 and samples consist of:

    Gene Colonies
    Bn PUM1 F2 1
    Mm DDX4 R2 1
    Mm PIWIL2 8
    Mm NANOS 8
    Mm PUM 5
    Mm TUDOR 3

    Mm PIWIL2 and Mm NANOS had positive colonies. Mm PUM, Mm TDRD1, and Bn PUM1 F2 were negative. Mm DDX4 R2 is probably not positive (less than 500bp).

    Colony check 2012-01-07 18hr 21min

     
  • Bruno Vellutini 13:45 on 2012/01/06 Permalink
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    PCR product check 


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    Since no colonies grew in the first try of the RACE PCR repeating Bugula genes and new Membranipora genes, I ran a product check with 2 µl of sample + 2 µl of loading dye; also including the repetition of the regular PCR from Membranipora. Products seem to be ok although Mm DDX4 R2 and Mm PIWIL1 R1 had very faint bands. Which strongly suggests, as before, that the problem lies on the ligation/transformation steps.

    PCR check for RACE and regular PCRs

     
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