The following protocol requires many tube changes. Be sure that each tube is clearly labeled with your sample number to avoid swapping samples with your partner. There are a few other steps you can take to avoid cross-contamination: switch pipet tips at every step, keep only one tube open at a time, and avoid getting liquids on the lip of any tube or column.
Before beginning this protocol, check the maximum spin speed of your centrifuge. Some centrifuges reach 20,000 g and others reach only 16,000 g, and the time for centrifugation will have to be adjusted proportionally. Note that rpm stands for rotations per minute while rcf stands for "relative centrifugal force." It is rcf that is equivalent to g-force, not rpm, because differently sized rotors will impart different forces at the same rotational speed.
- Obtain your 100 μL bird stool sample from the teaching bench ice bucket, according to the number you are assigned on today's Talk page.
- Work quickly and keep the sample on your ice bucket until you have finished adding the lysis reagent.
- Immediately add 1.4 mL of the lysis reagent (called buffer ASL) and vortex for ~ 1 min, until the solution is homogeneous.
- The fastest way to add the appropriate amount of ASL is to add 0.7 mL twice with your P1000; that way you don't have to rotate the pipet setting in between additions.
- A few insoluble particulates may remain. Try vortexing for another 20-30 sec interval up to four more times, and stop vortexing when the sample no longer visibly changes over that interval.
- Heat at 70 °C for 5 min on the heat block at the front bench.
- Vortex for 15 sec and centrifuge for 1 min at 20,000 rcf or 1.5 min at 16,000 rcf. Place your tubes so that weight is equally distributed in the centrifuge.
- Unfortunately, your centrifuges cannot be set for 1.25 min exactly.
- Transfer 1.2 mL of supernatant into a fresh 2 mL tube.
- Be sure to use the special 2 mL eppendorfs here and not the standard 1.5 mL eppendorf tubes.
- Hold the foil-covered InhibitEX tablet over the tube, and gently push until the tablet pierces through the foil and falls into the tube.
- Vortex until completely dissolved, which takes about 3 min for these samples.
- The solution will be homogeneous but somewhat thick.
- Incubate 1 min longer on a tube stand (with no shaking), and then centrifuge for 5 min (20K g) or 6 min (16K g).
- Transfer the supernatant (usually about 500 μL) to a 1.5 mL tube and again centrifuge 3 or 4 min as needed.
- In a fresh 1.5 mL tube, dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant above followed by 200 μL of buffer AL, pipetting to mix each time. Finally, add 15 μL chitinase.
- Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 2 hours.
Part 2: Lab practical
You and your partner may work together on the lab practical. (Note: this collaboration will not be the case for future quizzes.) You are of course welcome to give different answers should you disagree.
- Quick-spin to recover the part of the sample that has condensed in the eppendorf tube lid.
- To quick-spin, hold down the "short" button on your centrifuge for 3-5 seconds, then release.
- Add 200 μL of ethanol, mix by briefly vortexing, and transfer to a QIAamp spin column (atop its 2 mL collection tube).
- Be sure to label the column here -- not the tube -- with your sample number.
- Centrifuge for 1 min (1.5 min on slower centrifuges), and if necessary repeat the centrifugation until all the sample has gone through the column. (Unlikely in our case!)
- Move the spin column to a fresh collection tube, add 500 μL buffer AW1, and spin 1 or 1.5 min as needed.
- Move the spin column to a fresh collection tube, add 500 μL buffer AW2, and spin 3 or 4 min as needed.
- In the meantime, trim the cap off a fresh 1.5 mL eppendorf tube using small scissors that have been wiped down with 70% ethanol. Prepare a sticky label (in your team color) for the top: write the date and your sample identification number. You should also label the side of each tube, at least with short unique identifier, so you don't lose track of which sample is which in the following step.
- Move to yet another fresh collection tube and spin 1 or 1.5 min to rid residual buffer.
- This step completely removes remaining ethanol that could interfere with future reactions.
- Now transfer the column to your trimmed, well-labeled 1.5 mL tube and carefully pipet 150 μL buffer of AE onto the membrane.
- Incubate for 5 min, then spin for 1 or 1.5 min, cap, and store in your ice bucket. We will collect each sample and store it at -20 °C until next time.