Here’s hoping this is the last round of DNR sample preparation!

The plan for the week:

• Tuesday (that’s today!): Prepare tissue and sonicate with Grace
• Wednesday: BCA analysis all by myself
• Thursday: Mini-trypsin digestion with Jose and Kaitlyn
• Friday, Saturday and Sunday: Speed vacuum all samples. The speed vacuum can hold a maximum of 60 samples, so Laura, Jose and I all have to speed vacuum our samples separately.
• Monday: Desalt all samples
• July: Mass spec this shit :sunglasses:

This is what Grace and I did today:

Step 1: Label centrifuge tubes

• Tubes for the sample I would extract proteins from (“prot”)
• Tubes after sonication (“11 µL”)
• PRO TIP: Have “prot” and “11 µL” tubes be the same color so it’s easy to match them together

Step 2: Obtain samples for extraction from the -80ºC freezer.

Table 1. Proteins to extract, based on table from a previous lab notebook entry.

Site	Condition	1	2	3
PG	B	N/A	O51	O52
PG	E	O78	O56	O30
FB	B	O43	O40	O35
FB	E	N/A	O24	O49
WB	B	N/A	O121	O122
WB	E	N/A	O131	O144
SK	B	O99	O96	O113
SK	E	O106	O102	O91
CI	B	N/A	O21	O22
CI	E	O10	O06	O04

Step 3: Cut samples in half.

• 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

Step 4: 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

Step 5: Added 50 mM NH4HCO3 + 6M urea solution to samples

• Moved samples to wet ice. Samples remained in wet ice for the duration of the sonication process.
• Pipetted 100 µL 50 mM NH4HCO3 + 6M urea solution into one sample tube
• Repeated for all samples + 1 blank

Step 6: Homogenized samples

• An up-and-down motion worked better than a side-to-side motion (pounding > smushing)
• Homogenized as much as possible
• Resulting mixture was cloudy, difficult to see tissue fragments
• Repeated for all samples + 1 blank

Step 7: Vortexed samples

• 3 pulses on vortex set to speed 10 (maximum speed)
• Repeated for all samples + 1 blank

Step 8: Sonication preparation

• Filled containers with 1000 mL nanopure water each and labelled containers “1” and “2”
• Made an ethanol dry ice bath
• Filled a beaker with ethanol
• Added one piece of dry ice at a time
• Continually added ice over the course of sonication
• Ensured all samples needing sonication were in a wet ice bath
• Cleaned sonicator tip
• Dipped in ethanol for 5 seconds while on
• Dipped in first container of nanopure water (1) for 5 seconds while on
• Dipped in second container of nanopure water (2) for 5 seconds while on
• Wiped down with additional ethanol and a kim wipe

Step 9: Sonication

• Sonicated one sample
• Placed sonicator tip in one sample centrifuge tube for 10 seconds
• Immediately placed sample in ethanol dry ice bath for 5 seconds
• Moved sample to wet ice bath
• Cleaned sonicator tip using procedure explained above
• Repeated for the rest of the samples sequentially, and the blank
• Repeated sonication procedure for all samples and a blank 3 times total
• Sonication must proceed sequentially (one sample sonicated, then another sonicated)
• Cannot sonicate one sample immediately after it has already been sonicated
• MUST SONICATE REMAINING SAMPLES BEFORE RETURNING TO ONE SAMPLE

Step 10: Aliquot 11 µL of sample into a new tube

• Contents from “prot” tube would go into corresponding “11 µL” tube

Because the ethanol dry ice bath obscured some of the “prot” tube labels, Grace and I got mixed up when pipetting “prot” contents into the corresponding “11 µL” tube. Luckily, we were able to match them correctly! Having the “prot” and “11 µL” tubes be the same color really helped. O121, O99 and 049 now have 11 µL less protein than all of the other tubes.