Wednesday, Sept. 24, 2014

Finished extraction of WA13_1-12

  • RNase at 9:50a for an hour
Checked WA13_1-12 and OR2_1-12 on gel
  • WA13(scored from 1-5 on quality)
    1. 5
    2. 5
    3. 4
    4. 5
    5. 5
    6. 4
    7. 3.5
    8. 4
    9. 4
    10. 4.5(some deg)
    11. 4.5(some deg)
    12. 5
  • OR2_1-12
    1. 3.5
    2. 5
    3. 3.5
    4. 4
    5. 3.5
    6. 4
    7. 3(low conc)
    8. 3(low conc)
    9. 3(low conc)
    10. 5
    11. 5
    12. 5

Updated sample sheet here

Monday, Sept. 22, 2014

Started working in the Pritzker lab at the Field Museum.

  • Autoclaved 1 mL and 200 uL tips, razor blades, and 1.5 mL tubes
  • Reran 24 extractions (see 9/15)
    • 1 uL of DNA, 3 uL TE, 1 uL loading dye
    • Messed up .tiff export, but most extractions looked good
  • Set up 12 tissue digestions
    • OR2_1-12
    • 56 deg at 5:00pm

Sept. 12-13, 2014

Set up 12 re-extractions for testing out EconoSpin columns (and using RNase on 1st extractions)

  • Set up overnight tissue lysis at 4:50pm
    • CA6 1-3
    • WA1 1-3
    • CA4 1-3
    • BC4 1-3
      • not a redo…
Sept. 13
  • 5uL RNase at 11:40a (no mix during)
    • took off at 12:40p
  • switched collection tubes between AW1 and AW2 washes
  • Let AE buffer incubate for 3 min

Thurs. Sept. 11, 2014

DNA extractions

  • Set up 24 more samples for lysis
    • Humboldt Bay (best quality): CA6 7-12
    • N. Willapa (medium): WA1 7-12
    • Tomales Bay (poor): CA4 7-12
      • 10,11: ethanol, new tube, black/blue
    • Ladysmith (poor): BC4 16-21
  • Add 5 uL of 10 mg/mL RNase at 9:40pm
  • most tubes completely degraded
  • left until 10:40
  • CA6 11 required an extra 30 min of digestion and 2 uL more protienase A to fully digest
  • 1st centrifuge for 2 min instead of 1

Notes on RNAse Treatment (9/7/14)

From: http://seqanswers.com/forums/archive/index.php/t-17551.html)
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

Sunday, Sept. 7 2014

Sample storage
  • Finished checking all tubes to make sure tissue was covered in liquid at the bottom
    • 5 unlabeled tubes mixed between CA 5, 6, and WA O/P samples
  • replaced RNALater in BC pops with 80% ethanol
  • Ladysmith 10-17 and 18-25
    • frozen in blue capped tubes
    • 10-23
    • Stored in 80% ethanol
DNA extractions
  • Chose from 4 locations with variable storage quality
    • Humboldt Bay (best quality): CA6 1-6
    • N. Willapa (medium): WA1 1-6
    • Tomales Bay (poor): CA4 1-6
    • Ladysmith (poor): BC4 10-15
  • Left overnight (4:00pm) in 18.2 uL Proteinase K, mixing every 45 minutes