| || |
|-|background on DNA extraction |+|
| || |
sample choices -- bird species, time of year, cloacal vs stool maybe? |+|
background about -
time of , stool
| || |
Today you will begin your investigation of bacterial composition in different bird populations. Specifically, you will compare gulls from Cordova, Alaska with those that frequent a local reservoir (where exactly in MA?).
background about why this comparison might be of interest and how it fits into more cutting-edge research questions
Your starting point in lab will be an aliquot of bird stool suspended in a viral transport medium (VTM). The VTM helps preserve pathogenic viruses such as influenza for later study. The particular samples that we use today have been screened by the Runstadler lab and confirmed negative for influenza; as interesting as their research in avian flu is, we don't want to become unwitting participants!
To study the bacteria cohabiting with any given bird, we'll attempt to preferentially extract pathogen DNA (as opposed to mammalian DNA) from bird stool samples. We'll then amplify 16S ribosomal RNA gene sequences using the universal primers we discussed last time in a polymerase chain reaction (PCR). The PCR will result in a pool of different 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than a rarely present species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA (i.e., representing a single species of bacteria), thus allowing us to deconvolve our pool of DNA and analyze individual sequences. Briefly, we'll perform a second DNA extraction from several colonies per original bird stool sample and find out the sequence of each DNA through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.
Make overview schematic for lab progression to accompany above paragraph!
Returning to today's specific work, each of you will extract a DNA pool from a single bird stool sample using a QIAamp stool kit. The basis of this kit... chitinase modification and reasoning... etc.
Part 1: DNA extraction from bird stool, initiate
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 different 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; 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 3 min (20K g) or 4 min (16K g).
- Transfer the supernatant (usually about X mL) 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 10 μL chitinase.
- Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 2 hours [1.5??].
Part 2: Writing Across the Curriculum (WAC) session
During the enzymatic incubation, you will have an introductory session with our writing faculty.
Part 3: DNA extraction from bird stool, complete
- 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.
For next time
- QIAamp DNA Stool Mini Kit from Qiagen
- Chitinase from Sigma
- Stock solution prepared at X concentration in Y solvent
- Ethanol (200 proof)