Amplification of Bacterial Genomic DNA by Polymerase Chain Reaction to ID Soil Unculturable Flora
The genomic DNA extraction that we did in Lab2 has, no doubt, resulted in a mixed DNA population from a myriad of microorganisms as well as, probably, from plants, insects, or other life forms in the soil community. Since we are only interested in the scope of our bacterial population, we will amplify, by polymerase chain reaction, only bacterial DNA by using "universal" bacterial primers :a forward primer, Eub27F (5′–3′:AGA GTT TGA TCC TGG CTC AG) , and a reverse primer, Eub1492R (5′–3′: ACG GCT ACC TTG TTA CGA CTT). These primers are short sequences of single stranded DNA that are complementary in sequence to areas of the 16s rRNA gene. The 16S rRNA gene sequence is particularly good target gene for amplification because this gene (encoding a ribosomal subunit) contains conserved sequences of DNA common to all bacteria (to which the primers are directed) as well as divergent sequences unique to each species of bacteria (allowing identification of the bacterial species from sequence databases and sequence identifying software). Our "universal" primers will anneal to most bacterial DNA and initiate an amplification from the template DNA that begins with this common region, but ends, after 20 cycles of polymerase chain reaction in a thermal cycler, with a pcr product containing hundreds of unique copies of 16s rDNA, allowing identification of much of the bacterial flora present in the soil community, most of which is unculturable by conventional techniques.
Part A: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers
To review how the polymerase chain reaction works and how it exponentially amplifies specific sequences of DNA, go to the following web site:
All PCR reactions require a thermal cycler to elevate and reduce the reaction temperature quickly and keep it at a specific temperature for a prescribed amount of time. There is a basic pattern to these temp. cycles, but there are differences, so you must be sure to program the cycler with the correct time and temperature for your specific amplification. Traditionally, pcr used Taq polymerase, a heat stable DNA polymerase originally found in extremophilic, Thermus aquaticus living and reproducing in boiling hot springs. We are using a different polymerase, Finnzyme's Phusion High-Fidelity Polymerase, a proprietary reagent that uses a novel
Pyrococcus-like enzyme with a processivity-enhancing domain. Phusion DNA Polymerase generates long templates with an greater accuracy and speed than with Taq. The error rate of Phusion DNA Polymerase in Phusion HF Buffer is determined to be 4.4 x 10-7,
which is approximately 50-fold lower than that of Thermus aquaticus
DNA polymerase, and 6-fold lower than that of Pyrococcus furiosus, another proof-reading DNA polymerase.
Therefore, our pcr product DNA will have far fewer "mistakes" in the sequences that are replicated from template DNA. Our polymerase will also work much faster so our ~20 cycles will require less time than conventional Taq based pcr.
Protocol for PCR
Obain a tiny 0.2ml pcr tube from your instructor (choose the one prepared for your team in your team's color). All of the ingredients listed below in the table, except the template DNA, have been added together previously and kept on ice for you in these tubes.
Label it with a fine tipped Sharpie on the top and side with the code name for your sample. Do not use tape.
If your soil DNA isolate is at approximately 100ng/μL, you will follow the Template Table (shown below) adding 3μL DNAase free water and only 1μL of template DNA to the reagents that have already been premixed for you in your pcr tube (10μL master mix, 4μL DNAase free water, 1μL of each of 2 primers).
If your soil isolate DNA concentration was less than 20ng/μL, you will add 4 μL of DNA and no extra water. If your concentration was between 20 and 100ng/μL, calculate how much template DNA to add by using the formula 100 / your isolate's DNA conc. Add that number of microliters of DNA (not more than 4) and enough DNAase free water so that the number of microliters of DNA + microliters of water =4. Example: Your DNA conc. was 33ng/μL. 100/33 = 3.3 so you would add 3.3μL of DNA and 0.7μL of DNAase free water. Since your pcr tube already has 10μL master mix, 4μL DNAase free water, and 1μL of each of 2 primers, the total reaction volume for everyone will be 20μL.
It is very important to pipet these tiny volumes accurately. Use a P2 or P10 and the special small tips with a filter when pipetting DNA. Look at the tip after you draw up your measured volume to make sure you have liquid there.
Dispense the template DNA onto the side wall of the pcr tube close to the other liquid ingredents, watching to make sure that a small bead of liquid is left on the wall of the tube.
Without changing the tip, pipet up and down in the pcr mix to wash the tip and then wash some of the mixture over the bead of template DNA that may still be attached to the tube wall.
Tap the bottom of the tube (VERY GENTLY!) and flick the tube to mix. Do not treat these tubes roughly as they are quite thin-walled and can break or crack.
Bring your tube to your instructor; she will show you where the thermal cycler is located in E301. Your instructor will start the reaction when everyone's tubes are loaded.
|| amt. in a 20 μl
| Final Conc.
| 4 μL already in tube.
Want to achieve
total of 20 μl reaction vol.
Add from 0 - 4μl
| 2x Phusion Master Mix
|| 10 μl
| 27F primer
|| 0.5 μMolar
| 1492R primer
|| 0.5 μMolar
| template DNA
|| 1-4 μl
|| optimum is 100ng of DNA/reaction
The cycling program is shown below.
Thermal Cycler Program:
3 step program
| Cycle Step
|| # of Cycles
| Initial Denaturation
|| 5 min.
| 10 sec
| Final Extension
| 10 min
While the 16S rRNA genes from all of the bacterial species in your soil genomic isolate are being amplified in the thermal cycler, you will have about an hour to work on the soil community profiling assays that you started last week.
However, before you leave today, you will need to complete a "Clean-Up" of your pcr products (remove the unused dNPTs, primer dimers, salts, etc. The instructions for using a kit to purify your pcr products and get them ready for cloning next week are found later in this lab description in Part B. You will also need to set up a gel (described in Part C) to assess the purity of your pcr product and the success of your amplification.
Part B: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit
Notes before Starting:
Make sure 95% ethanol has been added to Buffer WS before first time use (see bottle label for volume).
All centrifuge steps are carried out at 17,900 x g (13,000) rpm) in a conventional tabletop microcentrifuge at roomtemperature.
1. Measure 500 μl of Buffer PX using your P1000 and add part of it to your thawed pcr product and the rest to a clean microfuge tube. Using your P200 set to 200 μL, remove all the pcrProduct/buffer mix in the pcr tube and add it to the PX buffer in the microfuge tube. Close the cap of the microfuge tube and mix.
2. Place a GenCatch™ spin column in a provided 2 ml collection tube.
3. Load no more than 700 μL of the pcr product/bufferPX mixture created in step 1 to the spin column and centrifuge for 60 sec.
4. Discard flow-through. Place the spin column back into the same collection tube.
(Collection tubes are re-used to reduce plastic waste.)
5. If you had more than 700 μL volume of pcrProduct/bufferPX made in step 1, apply the remaining volume to the spin column and centrifuge for 1 minute. Discard the flow through and place the spin column back in the same collection tube. If you applied all the pcrproduct to the spin column in step 3, skip this step and proceed to step 6.
6. Wash the spin column by adding 500 μL Buffer WF to the spin column and centrifuge for 60 sec. Be careful to use WF buffer!!
7. Discard flow-through and place the spin column back in the same collection tube.
8. Wash the column by applying 700 μL of Buffer WS to the spin column. Note that WS Buffer is different than the buffer used in step 6. Centrifuge the column for an additional 1 min. Discard the flow through
9. Centrifuge the spin column in the same collection tube at full speed for 3 more minutes to remove ethanol residue. It is crucially important to remove all ethanol residue; residual ethanol may inhibit subsequent enzymatic reactions.
10. Place each spin column into a new, clean 1.5 ml microcentrifuge tube (not a collection tube).
11. To elute DNA, add 50 μl of the Elution Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each spin column membrane. Let it stand for 2 minutes to allow it completely adsorb and then centrifuge the spin column in the microfuge tube for 1 min at 17,900 x g (13,000 rpm).
Keep your pcr product on ice until your instructor tells you that it's time to load the gel to determine the success of this amplification and clean-up.
IMPORTANT NOTES for using this kit: Ensure that the elution buffer is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. Store DNA at –20°C as DNA may degrade in the absence of a buffering
Part C: Agarose Gel Electrophoresis of Clean PCR PRODUCT
To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?
Your agarose gel is made of 1.0% agarose solution (w/v) in 1x TGE buffer (10x=0.25 Tris, 1.9M Glycine, 13mM EDTA) with SybrSafe™ stain.
DNA is uniformly negatively charged and will,therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA.
Procedure for Agarose Gel Electrophoresis of PCR products
Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.
You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 5 microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the loading dye by pipetting up and down before loading all 10 microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer with Sybr Safe DNA stain a proprietary reagent from Invitrogen used according to manufacturer's directions http://www.invitrogen.com) that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.
Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. 1x TGE buffer is used in this electrophoretic separation (25mM Tris, 0.19M glycine, 1.3mM EDTA. The gel will be run at 120V for approximately 30 minutes.
How will you judge a successful amplification? How many fragments and of what size do you expect to see?
Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate.
Make sure your pcr product is clearly labeled as pcr product and has your initials, team color, lab section (Tues or Wed), soil identifier code (T[tropical], CT[cool temperate], WT[warm temperate] and sample A or B (if there are two samples for a habitat) and the conc. of the DNA in ng/μL. In the next lab you will clone the fragments of 16s rDNA from the soil bacteria flora that are in your pcr product into a special genetically engineered cloning vector. That vector will be transformed into competent genetically engineered E. coli bacteria and they will be plated on selective media to find cells containing the 16s rDNA insert that we can send away for sequence analysis to determine the identity of some of the bacterial flora in your original soil sample.
While the amplification of bacterial 16srRNA genes goes on in the thermal cycler, we have about an hour to work on the community profiling analyses that we started last week.
In Lab3 you started some quantitative assessments of your soil communities' ability to digest cellulose, starch, and to solubilize phosphates---all important functional roles. Today while the thermal cycler completes our polymerase chain reactions, you will have about an hour to complete the colony colony counts from the differential media that you inoculated with dilute soil extract last week.
Community Soil Physiological Profiling PROTOCOL con't:
Examine the plates for processing of a particular nutrient (starch, cellulose or insoluble phosphates) in each differential culture medium. Remember that these differential media are not selective (they aren't designed to inhibit the growth of any groups of soil microorganisms) but they are differential media, in that they allow you to visibly SEE the difference in particular groups of microbes---in our cases, between those that produce and secrete a functional exoenzyme and those that don't. You will count the number of individual colonies showing a clear zone (halo) around the colony (using the plate with 30-300 total colonies) and compare those numbers with the number at the same soil dilution that grew on NA- a general purpose, non-differential medium.
1. Count the total number of colonies on the Nutrient Agar plate and assess total culturable CFUs.
2. Flood the starch plate with a thin layer of iodine and count the number of colonies that show starch digestion activity as a clear zone or non-blue halo around the colony).
3. Count the number of colonies that show cellulose digestion activity as a clear zone or halo around the colony.
4. Count the number of colonies (the colonies will be red) that show phosphate solubilizing activity as a clear zone or halo.
5. Use the soil extract dilution on the plates counted to calculate CFUs/gram of soil (wet weight) for each assessment medium. If you divide the number of colonies counted by the amount of inoculum plated times the dilution factor of that plate, you will obtain the number of cultivatable bacteria per gram of soil.
number CFU/dilution plated*dilution factor = number of CFU/gram
For example, if you counted 150 colonies on the 10-3 plate the calculation is:
150/(0.1ml plated*1X10-3dilution)= 150X104 which in scientific notation is written as 1.5X106 CFU/gram
6. Calculate the % of colonies positive for the enzymatic activity for each assay (# positive colonies/total count on nutrient agar X 100) in the original soil extract/ml. (For example: Once you calculate the total number of aerobically culturable bacteria (cfu/g) on the general purpose media, you can determine the % of the total number able to solubilize phosphate by dividing the number of phosphatase positive colonies by the total number of culturable colonies---if the colonies counted are from the same plate dilutions.)
1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.
2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.
3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.
4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.
5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.
6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.
7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.
8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.
9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.
10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.
11. Take off your lab coat and store it in the blue cabinet with your microscope.
12. Wash your hands.
Write a Materials and Methods Section
Some of the things we will stress in this lab, other than acquiring experience with the tools and techniques of the microbiologist, are analysis and presentation of experimental data and scientific writing. Our semester long project will end with a group presentation and an individually written final paper. The presentation will model a poster presentation given to community of peers at a scientific conference and the final paper will be written in the form of primary research report. The topic of both is Bacterial Diversity in Soil Communities: Functional Roles and Phylogenetic Relationships.
It can be a daunting task to write a single paper on several months of experiments, particularly when those experiments result in a huge amount of disparate data. To help you manage your project more effectively, and to prevent you from having an overwhelming amount of analysis and writing at the end of the semester, we have designed assignments throughout the semester that will be evaluated and graded. The feedback you receive on parts of the data analysis or on drafts of typically problematic sections of the paper, we hope, will help you work on the paper throughout the semester and help make the final submission exemplary.
Your first assignment in this process is to write up the protocols completed, so far, in Identification of the Bacterial Soil Community by 16s rRNA Gene Sequencing. That would include: acquiring a soil sample from a particular habitat, isolating genomic DNA from it, amplifying 16s rDNA by polymerase chain reaction (this amplification includes the adjustment of DNA concentration, the pcr product "cleanup" and the assessment of success by agarose gel electrophoresis). Although those procedures take up substantial space in the lab wiki, turning all those protocols and descriptions into a Materials and Methods section should require a page or less of text (double or 1.5 spaced). Remember that the Materials and Methods section of scientific paper does NOT read like protocol descriptions in the wiki. The emphasis is NOT on what you did in lab, step by step, in a time course description of your lab days, but rather on a BRIEF description of the process of turning starting material into whatever the goal material may be. In this case, the starting material is a particular soil sample and the goal is to end up with lots of double stranded DNA segments of a particular part of the 16s rRNA gene from as many of the bacteria found in your soil community as possible.
Each part of Methods & Methods in your final paper will be a separately titled section. The section titles should emphasize the experimental goal of that part of the larger experiment, rather than being a more general descripiton of procedures or equipment. Therefore, a good title for this Methods section would stress the amplification of _____ from________ by____, rather than "Pcr of_____. There may be titled sub-sections, or not, in each Materials and Methods section. Remember that the goal of a Methods description is to give a future investigator enough information to accomplish the same experimental goal, but, perhaps in a different context, scale, or using a different starting material. Therefore, you need to include all the essential information so that such a future investigator can make or buy all reagents and complete the experiment successfully to its goal, but you do not have to include details that an experienced investigator would infer (such as specifying that you "mix" after combining reagents or giving the type of general equipment such as the tube type you used). NEVER, EVER use the word "tube" in Materials in Methods. We often tell you, in a protocol, to do ____ to the "tubes". In the context of a lab manual procedure description, that's fine. For Materials & Methods, you must be more specific. It's what's happening to the materials in the tubes that is important. Tubes and plates are just pieces of plastic or glass. Your methods section should not read like a series of "did this, then did that" descriptions, but should, instead, describe how you acquired and processed the starting material to end up with the ending material.
Please look at the Methods sections of published scientific research papers (there are many in the References folder in your First Class lab conference) to see how the protocols in this wiki differ from Materials and Methods descriptions in a scientific paper. Notice that M&M can be very short, particularly if you have used a kit or a previously published protocol for a part or all of the experiment. If some part of your work is published elsewhere, such as in a journal article's methods section or in a kit manufacturer's website, you may reference the url of the kit manufacturer or use a journal article citation to the previously published directions. Use the citation format requested, EXACTLY!. Attention to detail matters. For BISC209, we are using the citation style of the journal Cell. It is given as (First Author's last name/ Year of publication) in the body of the paper AND there must be a FULL citation of the article in the References list at the end of the paper. If you use citations to previously published methods descriptions (and it is fine or desirable to do so), that means you MUST include a Reference page with the full citation. It is desirable to start creating such a cited References document now, so you won't have to search for the full citation for all your refrences later.
Since most of our protocols come directly the manufacturer of the main reagent or kit and others come from well-established published sources, writing the methods descriptions for this assignment should not be extensive in length. However, to write about how you accomplished your experimental goals succinctly and clearly, you must thoroughly understand them. It does not require thorough understanding to follow the directions in the wiki and end up with a successful experiment; however, to write about your experiments in such a way that someone who is not part of this course can understand what you did and how it contributed to your overall goal... that's is not so easy. If you make sure that everytime you leave lab you understand how each day's work fits into the project's overall goals, writing M&M, as well as the other parts of this paper, will be easily manageable. Don't wait until the end of the semester to figure out the big picture: what you are doing and why. If you want to keep practicing converting our protocols to M&M as you complete them, rather than limiting yourself to those we ask you to turn in for graded assignments, you will make the writing of your final paper much easier.
Please do NOT reference the wiki, as it not a primary source. Although you can ease the burden and length of this assignment greatly by referencing other journal articles or a reagent manufacturer's web site (such as the New England BIolab's website directions for using Phusion® High-Fidelity PCR Master Mix with HF Buffer-- the reagent we used to amplify our target DNA by pcr (found at: | http://www.neb.com/nebecomm/ManualFiles/manualF-531.pdf), the manufacturer does not include the sequences of the primers we used (since those are unique to your target DNA) nor the exact thermal cycler program (since that is tweaked for the length of the DNA section you want to copy and for the relative CG content of the template DNA). Therefore, you must give more than a brief citation and web address. In the case of the pcr amplification using a commercial master mix, you must be specific about the template DNA, the primer information for amplifying 16s rDNA, and give the exact thermal cycler program you used.
There is an extensive handout on writing a Methods section in the Resources section of this wiki. In this course we will use the citation style of the journal Cell, although it is only one of many scientific citation styles. The Wellesley Library has electronic access to Cell, so you can look at research reports in recent issues to see how the authors formatted their citations. Use those Reference pages as models when formatting your references. Attention to detail is crucial in scientific writing. You must follow the citation style requested exactly and uniformly.
This assignment is due at the BEGINNING of Lab 5. Do not come late to lab because you are printing or otherwise completing this assignment and you may NOT work on it during lab. There is a 5% per day late penalty for work for this course and since you have a week or more to complete assignments, illness (unless it is lengthy and serious) does not excuse you from the late penalty.
Continue monitoring and following the appropriate protocols to isolate the culturable bacteria.
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