Yaamini’s Notebook: DNR Sample Preparation Round 2

Slice and dice

Laura and I are going to start our proteomics pipeline with a second set of samples tomorrow, so today Grace and I prepped samples. Like last time, we had to split the samples we wanted in half.

Identified samples for protein extraction

Table 1. Samples for protein extraction, from this lab notebook entry. Originally I wanted to use O111 for extractions but I could not find it in the sample box. I used O117 instead.

Site Condition 1 2 3
PG B O25 O83 O54
PG E O31 O71 N/A
FB B O36 O70 N/A
FB E O64 O46 O32
WB B O129 O126 O135
WB E O140 O145 O147
SK B O117 O120 N/A
SK E O103 O101 N/A
CI B O11 O13 O16
CI E O01 O08 N/A

Labelled snaptop centrifuge tubes

  • Tubes for the sample I would extract proteins from (“1/2”)
  • Tubes after sonication (“11 µL”)

Cut samples in half

The reason for cutting each sample in half is to use one half for protein extractions, and another half for corresponding DNA extractions later on. Our protocol:

  • Obtained dry ice from Biochemistry J Wing
  • Placed sample centrifuge tubes (original vials and vials labelled “prot”) in dry ice to keep them from thawing
  • Created a 10% bleach solution (4 mL Clorox bleach in 40 mL nanopure water) to sanitize equipment
  • Obtained tweezers, weigh boats and razor blades
  • Placed weigh boat in dry ice to keep chill
  • Using tweezers, removed gill tissue from centrifuge tube and placed in weigh boat
  • Cut gill tissue in half with razor blade
  • Disposed of razor blade
  • Placed one gill tissue half back in original tube, the other in the tube labelled “1/2”
  • Put both tubes back in dry ice to stay cold
  • Tweezers cleaned by dipping in 10% bleach solution, and then rinsing in nanopure water
  • Repeated for all samples

When cutting the samples, I realized we forgot the step where we rinsed tweezers with nanopure AFTER we cleaned them with bleach! :open_mouth: I called Emma, and she said to dispose of those samples since they were possibly contaminated by bleach. Additionally, she said we should replace the bleach with ethanol, since trace elements of bleach in our samples would be problematic. Since I was dealing with all of my bare condition samples first, I replaced them with different samples.

Table 2. Revised samples for protein extraction. I wanted to use O127 for extractions but again, I couldn’t find it in the sample box. I replaced with O124.

Site Condition 1 2 3
PG B O26 O60 O90
PG E O31 O71 N/A
FB B O66 O39 N/A
FB E O64 O46 O32
WB B O128 O124 O137
WB E O140 O145 O147
SK B O118 O100 N/A
SK E O103 O101 N/A
CI B O12 O14 O17
CI E O01 O08 N/A

The revised protocol:

  • Using tweezers, removed gill tissue from centrifuge tube and placed in weigh boat
  • Cut gill tissue in half with razor blade
  • Disposed of razor blade
  • Placed one gill tissue half back in original tube, the other in the tube labelled “1/2”
  • Put both tubes back in dry ice to stay cold
  • Tweezers cleaned by dipping in ethanol, and then rinsing in nanopure water
  • Repeated for all samples

I noticed that the amount of gill tissue in the original DNR sample vials varied between samples. Some had large chunks of tissue that were easily split, some barely had any tissue. I ran into something similar last time, so I don’t think it’s going to be much of an issue.

Made 50 mM NH4HCO3 + 6M urea solution

Protocol used can be found here, or viewed below. This solution must be used no later than 24 hours after it is made, or it is no longer viable.

  • Measured 10 mL of nanopure water in a graduated cylinder, and poured into falcon tube
  • Weighed out 79.06 mg of ammonium bicarbonate (NH4HCO3) (0.0793g measured)
  • Added NH4HCO3 to falcon tube, vortexed until mixed
  • Weighed out 7.21g Urea (7.21g measured)
  • Added Urea to falcon tube, vortexed until mixed
  • Poured falcon tube contents into graduated cylinder
  • Topped of contents in graduated cylinder with nanopure water up to 20 mL
  • Poured contents of graduated cylinder into falcon tube

Checked amount of reagents in freezer

I double checked that we had at least 10 aliquots of DTT and IAA, and 2 aliquots of TCEP left in the freezer from Rhonda. All’s good to go for sonication tomorrow!

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