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    2bRAD Library 6 Larvae
    • Notes
    • Metadata
    • Relevant Items
    • 2bRAD Library Preparation
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    2bRAD Library 6 Larvae

    Tuesday, 1/10/2017Table1Thursday, 1/19/2017Saturday, 1/21/2017Friday, 2/17/2017Sunday, 2/19/2017Monday, 2/20/2017Tuesday, 2/21/201720170223_115119.jpg20170223_115134.jpgThursday, 2/23/2017
    Tuesday, 1/10/2017
    Thursday, 1/19/2017
    DNA extractions with E.Z.N.A Mollusc DNA Kit.
    ●
    Trouble getting larvae to settle out of RNALater. Had to take out at least 40 uL of RNALater from each tube and add more PBS buffer.
    ●
    Digested with ProtK for 45 minutes, add 5 more uL after 30 minutes.
    Saturday, 1/21/2017
    DNA quantification of 224s extracted on 1/19/17. Used 2 uL with HS buffer on Quibit unless otherwise stated.
    1.
    NF 7.27 224 (4): 91.7 ng/uL, used BR because concentration was too high for HS
    2.
    HC 7.27 (1): 6.88
    3.
    HC 8.5 (5): 38.9
    4.
    SS 8.5 (4): 55
    5.
    SS 7.27 (4): 55
    6.
    SS 7.29 (5): 52
    7.
    NF 8.10 (3 low): 19.8
    8.
    SS 8.10 (5): 38
    9.
    HC 7.30 (5): 49.2
    10.
    NF 7.30 (5): 9.48, used 10 uL with HS
    11.
    SS 7.30 (5): 46.5
    12.
    HC 7.29 (5): 54
    13.
    HC 8.10 (4.5): 36.1
    14.
    NF 7.29 (5): 53
    15.
    NF 8.5 (5): 36.1
    Ran out DNA on a .8% agarose gel. Quality values in (), 1(worst)-5(best)
    Friday, 2/17/2017
    DNA extraction of larvae used in Tile Set experiment and post-settlement oysters (spat). Using E.Z.N.A Mollusc DNA Kit.
    For larvae, I decanted as much RNALater as I could and then added ice cold PBS buffer so the larvae would sink. Then spun for 1 minute in centrifuge at 10,000.
    For spat, I carefully used tweezers to take out a subset (~75) oysters and put in a new tube.
    ●
    Larvae
    ○
    SS_SetB_20150810
    ○
    HC_SetB_20150810
    ○
    NF_SetB_20150810
    ●
    Spat
    ○
    SS_Set_20150827
    ○
    HC_Set_20150827
    ○
    NF_Set_20150827
    Quibit to get DNA concentrations. Added 600 ng all samples to 96-well plate and left with Airpore tape in 37degC incubator (3:00pm)
    Sunday, 2/19/2017
    9am- transfered plate to 4degC
    Monday, 2/20/2017
    ●
    Added 8 uL of nuclease-free water to wells
    ●
    then did digestion
    ●
    and ligation steps (see attached protocol).
    Tuesday, 2/21/2017
    ●
    Did PCR step of protocol.
    ○
    Based on a report from the UCHicago Genomics Core sequencing center, it looks like there is some over-PCR in my library prep, causing some fragments to form a "bubble" of 300bp long. So for this library, I cut down the PCR cycles from 19 to 17.
    ●
    Run out 2% TBE gel for samples and cut out.
    20170223_115119.jpg
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    Sample HC_224_20150727 did not seem to work. Sample SS_224_20150807 Had some bubble or something in the gel that halted half of the well from moving down. I just cut out the half that was at 170 bp. All other samples look great.
    20170223_115134.jpg
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    Sample NF_SetB_20150815_B run out on small gel, which took less time than I expected. Really hard to see ladder or gel (esp in this picture), so I had to guess a little where 170bp was.
    Thursday, 2/23/2017
    Finished gel extraction and Qubit of post gel-extracted samples. Combined 5ng of DNA from each sample, minus HC_224_20150727 as it had very low DNA conc and no band after PCR.
    Used Qiagen PCR cleanup kit on combined samples
    ●
    578uL total divided into 4 tubes (145uL per tube).
    ●
    725 added
    Dropped off at UChicago Genomics Core sequencing center.

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