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
    • Some issue with Xcode update won’t let me (
    • 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/ -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)

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


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
  • 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

Notes on RNAse Treatment (9/7/14)

We commonly use a variety of DNA sources. Classic proteinase K followed by phenol-chloroform extraction and ethanol precipitation. Qiagen Gentra Puregene (my favorite method). Qiagen DNeasy (manual or from Qiacube). Invitrogen Purelink.
Like you mentioned QC is the most important issue. Nanodrop to get the basic quality values. We expect 260/280 1.8-1.9 for RNase treated DNA (always RNase treat) and want 260/230 >1.9. Then use Qubit to get the concentration (best to dilute to ~250ng/ul) by nanodrop so sampling is accurate. Then run ~200ng on a 0.6-0.8% gel to check the size of the DNA. You should have a nice clean single band of high molecular weight DNA above 20kb and no DNA stuck in the well.
If you are only seeing 3-7% of nanodrop I’d want to ensure you have an RNA free prep and that it is not degraded. We have seen that degraded (smear on a gel) shows much lower concentration by qubit/picogreen than high molecular weight DNA at the same concentration as measured by nanodrop.
RNAse treatement
DNase contamination is the obvious reason for your reduced yield. You can either increase the temp to 60oC when the Rnase will be still active but not the DNase or you can include a small concentration of EDTA which will scanvenge the divalent ions helping the DNase.
DNase-free RNase A from Roche and Thermo
To 40 uL sample volume I add 2 uL of 2 mg/mL RNase A and incubate @ 37C for 10 min. This is prior to an ethanol precipitation.
RNA contamination can be removed by adding 2 microlitre of RNase A (10 mg/ml, Fermentas) to 20 microlitre of DNA dissolved in TE buffer (Tris–EDTA, pH = 8.0) and incubate for 3–4 h at 37 C.

*Decided to do RNAse treatment for 1 hour at 37degC after tissue lysis