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    Rerun 2bRAD Sequencing
    • Notes
    • Metadata
    • Relevant Items
    • 2bRAD Library Preparation
    Editing disabled on read-only entry.

     
    Rerun 2bRAD Sequencing

    Thursday, 5/19/201620160519_gel.jpgFriday, 6/3/2016Table1Tuesday, 1/10/2017Wednesday, 1/11/2017Monday, 1/16/2017Tuesday, 1/17/2017Thursday, 1/19/2017Saturday, 1/21/2017Monday, 1/23/2017Wednesday, 1/25/2017
    Thursday, 5/19/2016
    Setting up another 2bRAD library for Olympia oyster broodstock that were used in the Puget Sound common garden experiment.
    Ran a gel with 1 uL of DNA for the samples that failed 2bRAD sequencing, excluding those prepared by the Roberts lab. See analysis for read numbers per sample here:
    https://github.com/ksil91/2016_Notebook/blob/master/2brad%20BS%20analysis.ipynb
    1.
    'HC1-14': 3
    2.
    'HC1-2', (B): 3 (some deg)
    3.
    'HC2-11',: 2 (deg)
    4.
    HC2-15': 2.5 (deg)
    5.
    'HC2-5',: 3 (some deg)
    6.
    'HC2-6': 3.5 (some deg)
    7.
    'HC3-15': 2.5 (deg)
    8.
    'HC3-5': 3.5 (some deg)
    9.
    'HC4-17': 2 (deg)
    10.
    'HC4-19',: 2.5 (deg)
    11.
    HC5-2',: 2 (low)
    12.
    HC5-3': 2 (deg)
    13.
    'HC5-9',: 3 (some deg)
    14.
    'NF1-7': 2.5 (deg)
    15.
    'NF2-1': 4
    16.
    'NF3-12',: 1 (low, deg)
    17.
    NF3-2: 3.5
    18.
    'NF3-13': 2.5 (low)
    19.
    'NF3-14: 3.5 (low)
    20.
    'NF3-4: 2 (deg)
    21.
    'NF3-7: 4
    22.
    'NF4-18: 4
    23.
    'NF5-1', (B): 4
    24.
    'NF5-17: 2.5 (low, deg)
    25.
    'SS1-10: 4
    26.
    'SS1-11: 4
    27.
    'SS1-17: 3.5 (some deg)
    28.
    'SS1-20: 3.5 (some deg)
    29.
    'SS1-6: 3.5 (low)
    30.
    'SS1-7: 4
    31.
    'SS1-9: 4
    32.
    'SS2-10: 4
    33.
    'SS2-11: 2 (lo, deg)
    34.
    SS2-13: 4.5
    35.
    'SS2-5', (B): 2.5 (deg)
    36.
    'SS2-9: 4.5
    37.
    'SS3-18: 4
    38.
    'SS3-4: 4
    39.
    'SS4-5: 41
    40.
    SS5-8: 3 (bright, some smear)
    41.
    'SS5-10: 4
    42.
    'SS5-3: 5
    43.
    HC3-16: 3 (some deg)
    44.
    HC3-14: 3.5
    45.
    HC3-18: 4
    46.
    NF1-3: 2 (deg)
    47.
    NF2-7: 3.5 (some deg)
    48.
    NF2-11: 4
    49.
    SS1-18: 3.5 (low)
    50.
    SS3-5: 3 (low)
    51.
    NF2-19: 3.5
    52.
    NF2-10: 3.5 (low)
    ●
    Did not find: NF1-8, NF3-3
    ●
    Did not have enough wells to run: 'HC2-17', 'NF2-18B',NF2-19, 'SS3-5A', (A), 'NF1-21', 'NF2-10', 'NF5-16','NF4-15',
    ●
    Tubes were empty. Added 50 uL to each and then moved to new tubes. Did not run on gel:
    ○
    'SS2-14' , (empty), 'SS2-18' (empty)
    20160519_gel.jpg
    Loading...
    Loading...
    Extracted these samples using EZNA mollusc kit:
    ●
    SS2-14
    ●
    SS2-18
    Friday, 6/3/2016
    Pipetted out 900 ng of DNA. This spreadsheet will also be used to keep track of other metrics throughout the 2bRAD protocol for each sample.
    Tuesday, 1/10/2017
    ●
    Check on plate of samples.
    ○
    Some samples had very little liquid in them, likely due to evaporation. Added 200 ng DNA to these, as noted in the "Notes" section of above table.
    ●
    Qubit SS2-14 and SS2-18. Pipet out 900 ng
    ●
    Left samples in 37degC incubator with Airpore tape overnight.
    Wednesday, 1/11/2017
    Add 8 uL of NFW to samples and spun down. Moved samples to -20degC.
    Monday, 1/16/2017
    Digestion and Ligation step of adult redos. Mark Bitter from my lab is observing and helping out with this library prep as he may be using it on pooled mussel larvae samples.
    Made new aliquot of 150uM SAM.
    ●
    60 uL SAM plus 140 uL NFW.
    Tuesday, 1/17/2017
    Ran PCR of library finished on Monday.
    ●
    Barcode stock of BC2 is low- use other barcodes going forward.
    Thursday, 1/19/2017
    Run out gels of PCR and cut out. Started gel extraction.
    ●
    7.5 uL loading dye to PCR product
    ●
    Added 70 uL to each well. Used 2 extra wide rigs and 1 wide rig.
    ●
    Had Mark cut out some samples
    ●
    Added 40 uL of NFW to each tube and put in 4degC at 1:15pm
    Saturday, 1/21/2017
    Finished gel extraction of samples.
    ●
    Left in -80degC for ~1 hour (starting 11:15am)
    ●
    Spun down in refrigerated centrifuge at 4degC for 22 minutes (took 10 minutes to get down to 4degC).
    ●
    Pipetted out ~60 uL of liquid product into 2 96 well plates.
    ○
    Mark extracted samples H3-A4 into a separated 96 well plate.
    ●
    Sample G2 gel fell on table after pipetting out liquid.
    ●
    Put leftover gels and get-extracted samples in 4degC.
    Monday, 1/23/2017
    Quantify samples using high-sensitivity Qubit and pool. Put library in -20degC.
    Wednesday, 1/25/2017
    Drop off library at sequencing center. Tube labelled KS-2B-5. Barcode sample sheet can be found here.

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