Link to Lab notebook
https://www.evernote.com/pub/meganhintz/labnotebook

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2/26/2015
qPCR DNA extraction method trials continued

Created by: Megan Hintz

SAFS - Roberts Lab

Protienase K Digestion -

• Samples were mixed in the morning
• Turned the heat up to 95 C at 9:45 am after talking to Brent, overnight at 56 C should be enough - there is no need to have them continue for another 4-6 hours.
• The heat block reached 95 C at 10:10, cooked the samples at 95 C for 30 min and removed at 10:40

Nano Drop Reports

• Samples A & B spiked with Oly Larvae
• Samples A were prepared with DNAzol and samples B were prepared with Chelex

Report from Nano Drop & graphs available in evernote

qPCR -Protocol, calculations and plate setup available in evernote
Standards used - created from Port Gamble Larvae 160-180 µm stored in EtOH - the same larvae used to spike the samples for today prepared 7/30/14 and store in the fridge for a month, aliquoted out and froze on 8/22/14
Master Mix -SPUD A used at a concentration of -made calculations with 2 room for 2 extra positive controls to even out the amounts pipetted**Didn't have enough Oly primers, used fwd & rev Brent new about left over from Nate in the Friedman Lab: made new aliquots from the stock100 μL aliquot = 10 μL stock primer + 90 μL molecular H2OAlso made new aliquots of SPUD fwd, rev, and prope - 2 100μL of fwd and rev, 2 50μL of probeSPUD A 1:10^-10Procedure - order of adding chemicals to create master mix Started with the H2O - finished with Supermix

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2/25/2015

qPCR - DNA extraction methods trial - continued
SAFS - Roberts Lab

Continuing with the three extraction methods-DNAzol (Samples A)-Chelex (Samples B)-Proteinase K (Samples C)

Samples B & C were placed in the vacuum chamber to dry, after 4 hours they were completely dry.

Samples A (continued from yesterdays DNAzol extraction) 5. DNA Solubilization

• added 20 μL of nuclease free water

*Note: Sam advised letting the samples completely dry before resuspending the DNA in water, we are starting with a small amount of DNA (the invisible DNA pellet)

Chelex Extraction:

• Prepared a new 10% Chelex solution
• 40 mL prepared (weighed out 4.0 g of Chelex 100 and added to 40 mL of nano pure water)
• Added 0.5 mL of Chelex solution to each sample
• Heated at 95 C for 20 min
• I used the snap cap vials so I placed another heat block on top so they wouldn't pop oen
• inverted the samples a few times after 10 minutes to mix up the sample
• Each sample was spun down briefly and the supernatant was transfered into a clean vial labeled #B*

Proteinase K Digestion:

• Followed protocol (here)
• Added 0.5 mL of Proteinase K solution to each sample
• Placed the samples on the heat block at 56 C over night (placed on the heat block at 3:30 pm)

Checking samples prepared 2/23/15 with DNAzol

• Samples were prepared with DNAzol and not spikes with Oly larvae
• Checked for DNA presence using the Nano Drop
• No DNA was in the samples, the peak of the curves were at a wavelength of 230-240 not 260
• Report in Excel

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Lab Report: 2/24/2015 qPCR - DNA extraction methods

UW-SAFS: Roberts Lab

qPCR - DNA Extraction Trial: Comparing 3 different methods of DNA extraction
DNAzol, Proteinase K, and Chelex

Plan:

• Subsample samples from Oly Larval Trapping Samples 5/2-10/13 Left trap
• Take 3 20% subsamples from each vial for the three different extraction methods
• Spike subsamples with Oly Larvae
• Each samples was labeled with a reference number and labeled A, B, or C for extraction method

#A - DNAzol extraction#B - Chelex extraction#C - Proteinase K extraction
Subsampling procedures follow those used 2/23/15

1. Shake the sample vial until well mixed (~ 0.5 - 1 min)
2. Subsample 20% of the sample (3 mL) & transfer into a clean 15 mL
   1. Repeat 2 more times for 3 subsamples of each vial
3. Spike samples with Oly Larvae
   1. Oly Larvae from PSRF 7/16/14 stored in EtOH; Port Gamble 160-180 µm
   2. Subsamples 1-4 spiked with 1 Oly larva
      5-8 spiked with 5 Oly larvae
4. Centrifuge the samples at 3000 rpm for 1 min
5. Remove the supernatant from the top of the sample leaving ~ 1mL of EtOH and sample
   1. Set pipette to 750 µL and pipette out ~ 2 mL of EtOH off, using the pipette tip and remaining solution mix up the sample
   2. transfer to a 2 mL vial
   3. rinse the 15 mL vial with ~ 0.5 mL of EtOH and transfer into the 2 mL vial
6. Centrifuge the 2 mL vials at highest rpm for 3 min.
   (13.2 rpm)
7. Remove the majority of the excess EtOH from the sample by pipetting it off without disturbing the sample pellet
8. Samples are now ready for the next step in DNA extraction

Samples C left open to dry for p-K extractionSamples A & B - spun down briefly to and removed any excess EtOH
DNAzol Extraction - Samples A

1. Lysis/Homogenization
   1. added 0.5 mL DNAzol, vortexed for ~ 30 sec
2. Centrifugation
   1. centrifuged at highest rpm (13.2 rpm) for 10 min
   2. transfered resulting viscous supernatant into clean 2 mL vials
      transfered 0.5 mL - new vials labeled #A*
3. DNA Precipitation
   1. added 250 µL 100% EtOH into each sample, inverted 5-8 times to mix and let sit for 1-3 min
   2. centrifuged samples at 5,000 rfc for 5 min
   3. removed EtOH/DNAzol mixture without disturbing the invisible pellet of DNA
4. DNA Wash
   1. added 0.5 mL of 70% EtOH and inverted once
   2. centrifuged at 1,000 rfc for 2 min and removed EtOH by carefully pipetting, careful not to disturb the invisible pellet

Chelex Extraction - Samples B

• Nothing was done with these samples today

Proteinase K Extraction - Samples C

• Samples were left open in the fume hood for ~ 3 hours, then closed before leaving for the night - samples weren't dry

Real lab notebook page 1, page 2, page 3, page 4

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Lab Report: 2/23/2015 qPCR Subsampling trial
SAFS - Robert's Lab

Trial subsampling protocol with samples collected in "Oly larval trapping in Fidalgo 5/2-10/13 left trap"Samples were labeled 1-8
Subsample should be 20% of the original sample.

Plan:

1. Shake the sample vial until well mixed (~ 0.5 - 1 min)
2. Subsample 20% of the sample & transfer into a clean 15 mL
   (20% of the 15 mL vials = 3 mL)
   (20% of the 50 mL vials = 10 mL)
3. Centrifuge the samples at 3000 rpm for 1 min
4. Remove the supernatant from the top of the sample leaving ~ 1mL of EtOH and sample
5. Transfer the concentrated subsample to a 2 mL vial
   *some of the samples were clumped at the bottom and were clogging the pipette tip, for these samples I was able to pour some of the solution into the 2 mL vial
   1. transfer the solution by pipetting the solution into the new vial and then rinsing the 15 mL vial with ~ 0.5 mL of EtOH and pipetting that into the vial
6. Centrifuge the 2 mL vials at highest rpm for 3 min.
   (13.2 rpm)
7. Remove the majority of the excess EtOH from the sample by pipetting it off without disturbing the sample pellet
8. Samples are now ready for DNA extraction

Trial digestion - DNAzol: Genomic DNA Isolation Reagent Followed DNAzol method

1. Lysis/Homogenization
   1. Added 0.5 mL of DNAzol to each sample & vortex for 30 sec
2. Centrifugation
   1. Centrifuged at max rpm (13.3 rpm) for 10 min and transferred the resulting supernatant to a clean 2 mL vial
3. DNA Precipitation
   1. Added 250 μL of 100% ethanol to each sample
   2. DNA was not visible - each sample was centrifuged for 5 min at 5,000 rfc
   3. DNA pellet was not visible, removed ethanol/DNAzol mixture being careful not to disturb the invisible DNA pellet
4. DNA Wash
   1. Added 0.5 mL of 70% ethanol to each sample and inverted once
   2. Centrifuged again at 1,000 rfc for 2 min, removed as much of the solution as possible avoiding the invisible DNA pellet

Samples used today were from the first week of sample collection, they are from the extra left trap we collected.None of these samples were spiked with Oly larvaeI was unable to spool any DNA, there may not have been much DNA in the sample but continued with the method as a trial.
Real lab notebook page 1, page 2, page 3