Megan's+Notebook

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

2/26/2015 qPCR DNA extraction method trials continued

Created by: Megan Hintz

SAFS - Roberts Lab

Protienase K Digestion -  Nano Drop Reports  Report from Nano Drop & graphs available in evernote
 * 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
 * Samples A & B spiked with Oly Larvae
 * Samples A were prepared with DNAzol and samples B were prepared with Chelex

**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 stock 100 μL aliquot = 10 μL stock primer + 90 μL molecular H2O <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Also made new aliquots of SPUD fwd, rev, and prope - 2 100μL of fwd and rev, 2 50μL of probe <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">SPUD A 1:10^-10 <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Procedure - order of adding chemicals to create master mix <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;"> Started with the H2O - finished with Supermix

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif; font-size: 17.6000003814697px;">2/25/2015

qPCR - DNA extraction methods trial - continued <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;"><span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">SAFS - Roberts Lab

<span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Continuing with the three extraction methods-DNAzol (Samples A)-Chelex (Samples B)-Proteinase K (Samples C)

<span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Samples B & C were placed in the vacuum chamber to dry, after 4 hours they were completely dry.

<span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Samples A (continued from yesterdays DNAzol extraction) 5. DNA Solubilization <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">*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)
 * added 20 μL of nuclease free water

<span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Chelex Extraction: <span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px;">Proteinase K Digestion:
 * 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*


 * <range type="comment" id="542208094_1">Followed protocol ([|here])</range id="542208094_1">
 * 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)

<span style="display: block; font-family: Helvetica,Arial,'Droid Sans',sans-serif; font-size: 14px; line-height: 1.5;">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
 * <range type="comment" id="542270192_1">[|Report in Excel]

</range id="542270192_1">

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif; font-size: 17.6000003814697px;">Lab Report: 2/24/2015 qPCR - DNA extraction methods

UW-SAFS: Roberts Lab

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">qPCR - DNA Extraction Trial: Comparing 3 different methods of DNA extraction DNAzol, Proteinase K, and Chelex

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Plan: <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">#A - DNAzol extraction <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">#B - Chelex extraction <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">#C - Proteinase K extraction <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Subsampling procedures follow those used 2/23/15 <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> >> 5-8 spiked with 5 Oly larvae > (13.2 rpm) <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Samples C left open to dry for p-K extraction <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Samples A & B - spun down briefly to and removed any excess EtOH <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">DNAzol Extraction - Samples A <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> >> transfered 0.5 mL - new vials labeled #A*
 * 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
 * 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
 * 3) Repeat 2 more times for 3 subsamples of each vial
 * 4) **Spike samples with Oly Larvae**
 * 5) Oly Larvae from PSRF 7/16/14 stored in EtOH; Port Gamble 160-180 µm
 * 6) Subsamples 1-4 spiked with 1 Oly larva
 * 1) Centrifuge the samples at 3000 rpm for 1 min
 * 2) Remove the supernatant from the top of the sample leaving ~ 1mL of EtOH and sample
 * 3) Set pipette to 750 µL and pipette out ~ 2 mL of EtOH off, using the pipette tip and remaining solution mix up the sample
 * 4) transfer to a 2 mL vial
 * 5) 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.
 * 1) Remove the majority of the excess EtOH from the sample by pipetting it off without disturbing the sample pellet
 * 2) Samples are now ready for the next step in DNA extraction
 * 1) Lysis/Homogenization
 * 2) added 0.5 mL DNAzol, vortexed for ~ 30 sec
 * 3) Centrifugation
 * 4) centrifuged at highest rpm (13.2 rpm) for 10 min
 * 5) transfered resulting //viscous// supernatant into clean 2 mL vials
 * 1) DNA Precipitation
 * 2) added 250 µL 100% EtOH into each sample, inverted 5-8 times to mix and let sit for 1-3 min
 * 3) centrifuged samples at 5,000 rfc for 5 min
 * 4) removed EtOH/DNAzol mixture without disturbing the invisible pellet of DNA
 * 5) DNA Wash
 * 6) added 0.5 mL of 70% EtOH and inverted once
 * 7) centrifuged at 1,000 rfc for 2 min and removed EtOH by carefully pipetting, careful not to disturb the invisible pellet

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Chelex Extraction - Samples B <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">
 * Nothing was done with these samples today

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Proteinase K Extraction - Samples C <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">
 * 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, , ,

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif; font-size: 17.6000003814697px;">Lab Report: 2/23/2015 qPCR Subsampling trial <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">SAFS - Robert's Lab

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Trial subsampling protocol with samples collected in "Oly larval trapping in Fidalgo 5/2-10/13 left trap" <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Samples were labeled 1-8 <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Subsample should be 20% of the original sample.

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Plan: <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> > (20% of the 15 mL vials = 3 mL) > (20% of the 50 mL vials = 10 mL) > *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 > (13.2 rpm) <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;"> Trial digestion - DNAzol: Genomic DNA Isolation Reagent <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Followed
 * 1) Shake the sample vial until well mixed (~ 0.5 - 1 min)
 * 2) Subsample 20% of the sample & transfer into a clean 15 mL
 * 1) Centrifuge the samples at 3000 rpm for 1 min
 * 2) Remove the supernatant from the top of the sample leaving ~ 1mL of EtOH and sample
 * 3) Transfer the concentrated subsample to a 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
 * 2) Centrifuge the 2 mL vials at highest rpm for 3 min.
 * 1) Remove the majority of the excess EtOH from the sample by pipetting it off without disturbing the sample pellet
 * 2) Samples are now ready for DNA extraction
 * 1) Lysis/Homogenization
 * 2) Added 0.5 mL of DNAzol to each sample & vortex for 30 sec
 * 3) Centrifugation
 * 4) Centrifuged at max rpm (13.3 rpm) for 10 min and transferred the resulting supernatant to a clean 2 mL vial
 * 5) DNA Precipitation
 * 6) Added 250 μL of 100% ethanol to each sample
 * 7) DNA was not visible - each sample was centrifuged for 5 min at 5,000 rfc
 * 8) DNA pellet was not visible, removed ethanol/DNAzol mixture being careful not to disturb the invisible DNA pellet
 * 9) DNA Wash
 * 10) Added 0.5 mL of 70% ethanol to each sample and inverted once
 * 11) Centrifuged again at 1,000 rfc for 2 min, removed as much of the solution as possible avoiding the invisible DNA pellet

<span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Samples used today were from the first week of sample collection, they are from the extra left trap we collected. <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">None of these samples were spiked with Oly larvae <span style="background-color: #ffffff; color: #262626; display: block; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">I 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, ,