Tues. Nov. 4, 2014

  • Finished extraction from Nov 3.
    • RNAse 11:30-12:40
      • used new RNAse halfway through, same conc
    • Eluted half in dH2O to see if difference with PCR
      • CA1_1,2,14,18,20,17,13,12
      • BC3_5,8,3,6
    • CA1_17 <->CA1_15
  • Ran out extractions on gel (BC3_01-9,11; CA1_1,2,4,5, 13-22)
    • BC3_1: 4
    • 2: 5
    • 3: 4 (some smear)
    • 4: 4
    • 5: 4
    • 6: 3.5
    • 7: 5
    • 8: 4.5
    • 9: 5
    • 11: 4.5
    • CA1_1: 4(very bright, some smear)
    • 2: 5
    • 3: 4
    • 4: 5
    • 13: 0
    • 14: 0
    • 15: 0
    • 16: 0
      • Ladder not visible either, redo
    • 17: 5
    • 18: 4
    • 19: 4
    • 20: 4
    • 21: 5
    • 22: 5
  • Setup PCR (36):
    • 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

PCR protocol

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My attempts at In Silico Digestion

  1. Installed NumPy
    • Issue setting PYTHONPATH permanently for IDLE. Changed.bash_profile and restarted computer, but still only works in terminal.
  2. Attempted to install MySQLdb…
  3. Set up Enthought Canopy (BioPython not supported for free)
  4. Tried to Install Biopython http://biopython.org/DIST/docs/install/Installation.pdf
    • Some issue with Xcode update won’t let me (https://www.biostars.org/p/99125/)
    • building ‘Bio.cpairwise2’ extension
      Compiling with an SDK that doesn’t seem to exist: /Developer/SDKs/MacOSX10.6.sdk
      Please check your Xcode installation
      gcc -DNDEBUG -g -O3 -arch x86_64 -isysroot /Developer/SDKs/MacOSX10.6.sdk Qunused-arguments -Qunused-arguments -Qunused-arguments -I/Applications/Canopy.app/appdata/canopy-1.4.1.1975.macosx-x86_64/Canopy.app/Contents/include/python2.7 -c Bio/cpairwise2module.c -o build/temp.macosx-10.6-x86_64-2.7/Bio/cpairwise2module.o
      clang: error: no such file or directory: ‘Qunused-arguments’
      clang: warning: no such sysroot directory: ‘/Developer/SDKs/MacOSX10.6.sdk’
      error: command ‘gcc’ failed with exit status 1
  5. Ended up getting an Ubuntu virtual hardrive and getting Biopython on there
Retry (11/5/14)

Monday, Nov. 3, 2014

  • Run out PCR from 10/22
    • All bright bands at 500 bp, control=negative
  • Rerun DNA extracts
    • BC2_13: very low conc
    • CA4_17: vey bright with smear
    • CA4_19: 5
    • OR2_17: very low conc
    • BC1_3: nothing
    • BC1_6: nothing
  • Set up overnight extraction BC3_01-9,11 and (San Diego) CA1_1,2,4,5, 13-22
    • BC3_10 was dark, cloudy so did not extract
    • BC3_2 and 7 had yellow ethanol, put in new tubes/ethanol
    • BC3_8 had very little tissue (juvenile)

Updated sample sheet here

Tues, Oct. 21, 2014

Checked extraction done on 10/15
  • BC2
    • 13: 3 (low conc, may have washed out)
    • 14: 5
    • 15: 4.5 (some smearing)
    • 16: 5
    • 17: 4(smearing)
    • 18: 5
    • 19: 5
    • 20: 5
  • CA4
    • 13: 5
    • 14: 4.5
    • 15: 4(very bright, some smearing)
    • 16: 5
    • 17: 3(very bright, very smeared, redo)
    • 18: 4
    • 19: 3(very bright, very smeared, redo)
    • 20: 4.5
  • OR2
    • 13: 5
    • 14: 4
    • 15: 4
    • 16: 4
    • 17: 3.5 (may have washed out)
    • 18: 4.5
    • 19: 5
    • 20: 4

Updated sample sheet here

Protocol for 16S PCR, SAP/Exo, Sequencing Reaction

Dilute primers to 100 uM by adding 10x # of nmol H2O
Working stock (5 uM): 2.5uL stock, 47.5uL dH2O
PCR
MM1
Comp
1x Volume
40x
Final Conc
Water
6 μl
240
dNTPs
.5 μl
20
200 μM
R primer
2.5  μl
100
0.5 μM
F primer
2.5 μl
100
0.5 μl
MM2
Water
9.17 μl
366.8
10x buffer
3.2 μl
128
2 mL MgCl2
Taq
.13 μl
5.2
1.25 U/rxn
Add 1 μl DNA to each tube
Step
Cycles
Time
Temp
Initial Denaturation
1
2 min
94 deg
Denaturation
Annealing
Elongation
1-8
30 s
1 min
1 min
94
54,53,52,51,50,49,48,48
72
Denaturation
Annealing
Elongation
9-33
30 s
1
1
94
48
72
Final Elongation
1
7
72
forever
10
SAP/Exo
MM
Reagent
1x
13x
Sap
0.5 ul
6.5
Exo
0.5 ul
6.5
5 ul of PCR product + 1uL MM
Step
Time
Temp
Incubate
30 min
37 C
Denature
15 min
80 C
Chill
forever
25 C
Sequencing

Wed. Oct. 15, 2014

Finish extractions of OR2, BC2, and CA4
  • BC2_13-20 at 56deg 10:00pm
    • BC2_14 new ethanol (empty)
  • OR2_13-20 56deg at 10:20pm
    • OR2_16 new tube/ethanol (dirty)
  • CA4_13-20 56deg at 10:50p
    • replaced ethanol for CA4_14, 17(dark), 19 (dark, cloudy)
      • Somehow have 2 14s and no 13 after extraction.
  • Used lab proteinase K bc mine was missing (stolen??). Kevin recommended 15 uL
  • 5:50p -> RNase added, 37deg

Plan for identifying if some samples are Pacific oysters

  1. Finish OR2 (13-20), BC2(13-20), and CA4 (13-20)
  2. Do 16S verification while finishing extractions
    1. 1st sequencing run:
  1. CA4_1
  2. CA4_2
  3. CA4_3
  4. CA4_7
  5. CA4_8
  6. CA4_9
  7. CA4_11
  8. CA4_12
  9. CA4_13
  10. CA4_14
  11. CA1_1
  12. CA1_2
  13. CA1_13
  14. CA1_14
  15. BC3_1
  16. BC3_2
  17. BC3_3
  18. BC3_4
  19. BC3_5
  20. BC3_6
  21. BC3_7
  22. BC3_8
  23. BC3_9
  24. BC3_11
  25. BC1_1
  26. BC1_2
  27. BC1_4
  28. BC1_5
  29. BC1_7
  30. BC1_8
  31. BC1_9
  32. BC1_10
  33. BC1_11
  34. BC1_12
  35. BC2_1
  36. BC2_2
  37. BC2_3
  38. BC2_4
  39. BC2_5
  40. BC2_6
  41. BC2_7
  42. BC2_8
  43. BC2_13
  44. BC2_14
  45. OR2_1
  46. OR2_2
  47. OR2_13
  48. OR2_14

Polson et al. (2009) paper using 16S sequencing for taxonomic identification of O. lurida

OstreaTaxonomy

Wednesday Oct. 1, 2014

Extraction of WAO9_1-12

  • 56deg at 10:45a
  • Tissue was often dried or not fresh looking; changed RNALater for 85% ethanol in some cases (updated on sample sheet)
    • 1, 12 esp
  • Finished extraction of WA9O_1-12
Check extractions of BC1_1-12 and BC2_1-12
snapshot-29A51ABA-3AC8-4C2B-ABA9-8C59C4299065
  • BC1_1-12
    1. 4.5
    2. 4.5
    3. 2 (possibly low conc?)
    4. 3
    5. 4.5
    6. 2.5(low conc, no smear)
    7. 2.5
    8. 3
    9. 4
    10. 4
    11. 4.5
    12. 4.5(bright, possible smear)
  • BC2_1-12
    1. 4.5
    2. 4
    3. 3.5(some smear)
    4. 3.5
    5. 3.5(smear)
    6. 4.5
    7. 3.5
    8. 3
    9. 3.5(some smear)
    10. 4
    11. 3.5(smear)
    12. 5
Updated sample sheet here

Monday Sept. 29, 2014

Set up 24 samples for extraction

  • BC1_1-6, 8-12
    • BC4_7 (see below)
  • BC2_1-12
  • Did dissections in back room hood; thawed 6 at a time
    • BC1_1-6: 11:05a
    • BC1_7-12: 11:25a
    • BC2_1-6: 11:44a
    • 12:10p
  • There are 2 BC4_7 labelled tissues, likely that this one is actually BC1_7 but will discard just in case
  • Double check main tissue of BC2_12-> lysis buffer contamination possible
Finished extractions
  • 7:00p RNase at 37deg

Updated sample sheet here