Wed. Dec. 3, 2014

Extract

  • BC3_12,13,15,16,17,18
  • WA12D_1,3,4,5
  • WA10T_1,2,4,5,6,7

Run out gels

  • CA7_1-12
    • 1: 4
      2: 5
      3: 3.5(low)
      4: 4
      5: 3.5 (low)
      6: 0
      7: 3(low)
      8: 3
      9: 3
      10: 2(low)
      11: 2(low)
      12: 2(low)
    • redo CA7_6
  • OR3_1,2,3,5,6,9,10,12,13,15,17,18
    • 1:4.5(bright, some smear)
      2: 4
      3: 4.5(bright, some smear)
      5:5
      6: 5
      9:5
      10:5
      12:5
      13:5
      15:5
      17:5
      18:5
  • WA9_1-12
    • 1:1(deg)
      2:0
      3:0
      4:4
      5:5
      6: 2.5(low)
      7:0
      8:2(low)
      9:2.5(low)
      10:2(low)
      11:3(low)
      12:2.5(low)
    • redo WA9_2,3,7
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Tuesday Dec. 2, 2014

Extract DNA from CA2_1,2/6,3,4,5,7, and CA3_1,2,3,4,5,6

  • Column lids snapped off in centrifuge for 2,6 -> threw away. Need to reextract either 2 or 6

Gel of DNA extracts:

  • CA6_1,2,3,7,8,9,10,11,12
    • 2: 3.5
      10:3.5
      11:3
      12:3.5
    • need to redo CA6_1,3,7,8,9OR1_1-12
  •  OR1_1-12
    • 3: 3
    • 4: 3
    • 5: 5
    • 6:4
    • 7:5
    • 8:5
    • 9:5
    • 10:4.5
    • 11:5
    • 12: 5
    • redo OR1_1,2
  • BC4_1,2,16,18,19
    • all still low
  • CA4_1,2,3,7,8,9,11,12
    • 1: 3
      2: 4
      3: 1.5
      7:4
      8:5
      9:5
      11: 4.5
      12:5
  • WA1_1,2,3,7,8,9,10,11,12
    • 1: 5
      2: 5
      3: 4.5
      7:5
      8: 5
      9: 5
      10: 4.5
      11: 4.5
      12: 5

Sequencing Results from Nov. 22

Crassostrea gigas:

  • BC3_6
  • BC3_5
  • BC3_8
  • all from Harris Point, Vancouver Island (of course they’d be from the site that cost $300 extra to go to)

Ostrea

  • BC2
    • 1
    • 2
    • 3
    • 4
    • 5
    • 6
    • BC2_7
    • 8
    • 13
    • 14
  • BC1
    • 1
    • 4
    • 2
    • 5
    • 7
    • 8
    • 9
    • 10
    • BC1_11
    • BC1_12
  • BC3
    • 1
    • 2
    • BC3_3
    • 4
    • 9
  • CA4
    • 1
    • 2
    • 3
    • 7
    • CA4_8
    • 9
    • 11
    • 12
    • 13
    • 14
  • OR2
    • 1
    • 2
    • OR2_13
    • 14

Sequences were too low of quality (b indicates reverse)

  • BC1_1b
  • BC2_13b
  • BC2_14b
  • BC2_1b
  • BC2_2b
  • BC2_8b
  • BC3_11a and b
  • BC3_7ab
  • CA4_11b
  • CA4_13b
  • CA4_14b

Realized I didn’t need to do forward and reverse because sequences are so different between Crassostrea and Ostrea that I do not need to confidently call every single nucleotide.

Sat. Nov. 22, 2014

Extract CA7_1-12
  • 56deg at 10:40a
  • RNase at 5:30p
Finish extraction of OR3_1,2,3,5,6,9,10,12,13, 15,17,18
  • -Put stock tissues in new tubes/ethanol: 11,8,1,16
  • 37 deg at 10:00a

Sequencing run

  • Sequencer dropped the plate at beginning of run, had to restart the system. Tapped sequencing plate on table to settle reactions back at bottom of well.

11_20-Seq-Run

Nov. 21, 2014

Run out extractions of BC4_1,2,3,14,15,16,18,19,20,21, CA5_5-16

  • BC4
    • could not find BC4_13
    • 1: 2.0
      2: 2.5
      3: 3.0
      13: 2.0 (no RNAse)
      14: 2.0 (no RNase)
      15: 0.0 (no RNAse)
      16: 1.0
      18: 0
      19: 3(low)
      20: 3.5
      21: 4.0
    • Redo gel of BC4_1,2,16,18,19
  • CA5
      • 5: 4.0
                -6: 4.0
                -7: 3.5
                -8: 4.0
                -9: 3(deg)
                -10: 3.0
                -11: 3.5
                -12: 3.0
                -13: 3.5(low)
                -14: 3.5(low)
                -15: 4.5
                -16: 5.0

Gel 11_21_14

Start extraction of OR3_1,2,3,5,6,9,10,12,13, 15,17,18 and CA7_1-12 to leave digesting over night

Monday Nov. 17, 2014

Run out test digestion on gel, 1 uL against 1 uL DNA
  • looked digested, but will run out 5uL of digested DNA for clearer gel pic
Purify Sequencing reactions
  • EtOH/EDTA precip of 86 samples following Pritzker protocol
  • Did not observe pellet in wells
  • After step 11, left in 65 deg oven for 5 minutes to ensure etOH evaporated, then wrapped in foil and left in 4deg C fridge

Thursday, Nov. 13, 2014

In order to determine if ApeKI restriction enzyme will work for O. lurida, did test digestion with 4 individuals from different populations. Used ApeKI from Winston.

Test digestion with ApeKI

  • Determine concentration with Qubit (did not update standard from 11/5))
    • OR1_2: 4.78 ng/ul
    • W13_1: 6.82
    • BC4_17: 9.62
    • CA1_17: 96.4
  • Dry down in CentriVac at 60, ul for 200 ng:
    • B: 20.79
    • C: 2.07
    • O: 41.84
    • W: 29.32
  • ApeKI digestion at 75deg
    • 17 ul dH2O, 2 ul NEB3, 1ul ApeKI
    • 2:10p until 4:15pm
Sequencing Reaction of Sap/Exo
  • 86 reactions
    • well 16 from SapExo was empty (did not put lid on tight enough?)
      • BC3_11
  • Used cyc-seq program from Barber

Tuesday, Nov. 11, 2014

SAPExo of 44 PCR reactions

  • CA4_1
  • CA4_2
  • CA4_3
  • CA4_7
  • CA4_8
  • CA4_9
  • BC3_1
  • BC3_2
  • BC3_3
  • BC3_4
  • BC3_5
  • BC3_6
  • BC3_7
  • BC3_8
  • BC3_9
  • BC3_11
  • BC1_1
  • BC1_2
  • BC1_4
  • BC1_5
  • BC1_7
  • BC1_8
  • BC1_9
  • BC1_10
  • BC1_11
  • BC1_12
  • BC2_3
  • BC2_4
  • BC2_5
  • BC2_6
  • BC2_7
  • BC2_8
  • BC2_1
  • BC2_2
  • BC2_13
  • BC2_14
  • CA4_11 (9/11)
  • CA4_12(9/11)
  • CA4_13
  • CA4_14
  • OR2_1
  • OR2_2
  • OR2_13
  • OR2_14

Autoclave new tubes and 200 ul tips