BISC209: Lab3
Lab 3: Continue Soil Microbial Communities & Diversity Project
Today you will continue your identification of the total bacteria in your soil habitat by measuring the concentration of the genomic DNA that you isolated in Lab 2. When your DNA is at the proper concentration (100ng/μL) you will amplify only the 16S rRNA gene from hundreds or thousands of the different species of bacteria (some of which are culturable and most of which are non-culturable) using "universal" bacterial primers and a proof-reading DNA polymerase. Once our pcr has had time to go through enough cycles to amplify the 16S rDNA, we will remove the excess dNPTs, primer dimers, etc. from the pcr products by performing a "clean-up" procedure. In the Lab 4 you will clone your 16s DNA into a special cloning vector and start the transformation of those plasmids into gentically modified E. coli bacteria. We will plate the bacteria on selective media so that the bacteria that took up a cloning vector with an antibiotic resistance gene (transformants) will grow and the bacteria that did not take up a plasmid containing your pcr product DNA and the resistance gene (non-transformed bacteria) will not. We hope that we have hundreds of well isolated colonies from the transformation and selection process. In Lab 5, we will choose 96 if those bacteria, each containing an inserted 16S rRNA gene from (we hope) many different bacterial species from your soil samples, and get them ready to send away, as glycerol stocks, to a company that will sequence the 16S gene from the cloning vector (not the E coli's 16S gene). We should get our sequencing data back within two weeks of mailing off our clones. At that point, after having a tutorial session on how to analysis the sequencing data, we will be able to identify many of the bacteria in our soil habitat that we could not identify by traditional culture techniques.
Part A:Measuring the Concentration of DNA
There are two Nanodroppers in the BISC Equipment room, L308. Both the ThermoScientific NanoDrop 2000 and The NanoDrop ND-1000 Spectrophotometers measure DNA by taking Absorbance at A260nm. These spectrophotometers use only 1 microliter of sample and do not require cuvettes. The sample is held in place by fiber optic technology and surface tension that holds the sample in place between two optical surfaces that define the pathlength vertically and dynamically. Measurement can be assessed in a range of 2 to 3700nm/microliter dsDNA. These are expensive machines so make sure you follow the directions carefully and ask your instructor for guidance as needed.
More information is available from the manufacturer's website at: | http://www.nanodrop.com/HowItWorks.aspx
Using the Nanodroppers
1. Clean the upper and lower optical surfaces of the sample retension device by pipetting 2 microliters of clean deionized water onto the lower optical surface. Close the lever arm and tap it a few times to bathe the upper optical surface. Lift the lever arm and wipe off both optical surfaces with a Kimwipe.
2. Open the NanoDrop software from the Desktop of the computer and select the nucleic acids module.
3. Initialize the machine by placing 1 microliter of clean deionized water onto the lower optic surface, lower the lever arm, and select initialize from the NanoDrop software. Once initialization is complete (~10sec.), clean both optical surfaces with a Kimwipe.
4. Perform a blank measurement by loading 1 microliter of Solution 6 (10mM Tris) and select Blank. Note that this blanking step may use something other than Tris depending on what your sample is dissolve in. Often the blank will be deionized water if you have concentrated your DNA sample already with the ethanol precipation and resolubilized it in water.
Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water will be fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.
5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample.
You should ensure that the appropriate constant (50 for dsDNA or 40 for RNA) has been chosen. The software automatically calculates the nucleic acid concentration. If the calculation is done by hand, the A260nm is represented as a 1cm path for convenience, even though 1-nm and 0.2nm paths are actually used during the measurement cycle.
Clean Up
When the last sample was been measured, clean the sampling device by repeating step 1.
To Dilute or concentrate your DNA for pcr amplification of 16S rDNA
The final volume of eluted DNA from the DNA isolation procedure should be ~100μL. We want our sample to be at a concentration of ~ 100ng/μL for our pcr amplification. Do you need to dilute or to concentrate your DNA isolate?
Diluting the DNA
If your isolate is more concentrated than 100ng/μL, you can dilute it in sterile water or 10mM Tris (solution 6). Ask your instructor which is preferred. To calculate how to dilute your DNA, you will use the formula V1 x C1 = V2 X C2, where V1 is ~100μL volume of your eluted DNA from the PowerSoil isolation, C1 is the DNA concentration you obtained from the Nanodroper, C2 is 100ng/μL (the conc. you desire), and V2 is what you will solve for. Please check with your instructor after you have made this calculation and BEFORE you dilute your DNA.
Concentrating the DNA:
The DNA may be concentrated by adding 4 microliters of 5 M NaCl to your ~100μL DNA isolate and inverting 3-5 times to mix. Next, add 200μL of 100% cold ethanol and invert 3-5 times to mix.
Centrifuge at 10,000 x g for 5 minutes at room temperature. Decant every bit of the liquid without taking any of your pellet, using your P200. The pellet must be completely dry and every trace of ethanol must be removed. Remove residual ethanol in a speed vac, dessicator, or air dry. While your sample is in the speed vac calculate the volume of water you will need to resolubilize your DNA to the appropriate concentration. Check your calculations with your instructor BEFORE you add any water to your dried pellet of DNA. Use the next paragraph for how to calculate the dilution.
Use V1 x C1 = V2 x C2 to determine how much water you will eventually add to your dry DNA pellet. Remember that you know the original concentration, C1, from the nanodrop measurement and you know the C2, 100ng/μL, the concentration you desire. You know V1, is ~1 since you have essentially no volume to your pelleted DNA. You want to solve for V2, which is how much volume you desire to give your 100ng/μL.
Be sure that you ask your instructor to check your calculation before you go on the next step.
Resolubilize your precipitated DNA in the calculated volume of sterile water. After adding the appropriate volume of diluent to your tube, vortex vigorously at highest speed for 2 full minutes to make sure your DNA is completely in solution.
When your DNA is at the appropriate concentration, 100 nanograms/μL,
proceed to the next step: Amplification of 16s rDNA by Polymerase Chain Reaction.
Amplification of Bacterial Genomic DNA by Polymerase Chain Reaction to ID Soil Unculturable Flora
Our genomic DNA isolation has, no doubt, resulted in a mixed DNA population from a myriad of microorganisms as well as, probably, some contaminant DNA from plants, insects, or other life forms in the soil community. Since we are only interested in the scope of our bacterial population in this study, 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 rDNA gene. The 16S rDNA 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 35 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 B: PCR Amplification of 16s rDNA 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:
PCR animation
http://www.dnalc.org/resources/animations/pcr.html
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 archae bacteria, Thermus acquaticus 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. 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 0.5ml pcr tube from your instructor (choose the one prepared for your team in your team's color). Label it with a fine tipped Sharpie on the top and side with the code name for your sample. All of the ingredients below, except the template DNA, have been added together already and kept on ice for you. You must add 1 microliter of your template DNA that has been returned to you frozen. This is a tiny amount and you must make sure that you get it all into the tube. Use a P2 or P10 and the special small tips and look at the tip to make sure you have something there when you have drawn up your 1 microliter. 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. 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 who will show you where the thermal cycler is located and start the reaction when everyone's tubes are loaded. The cycling program is shown below. While the 16S rDNA is being amplified in the thermal cycler, you will proceed with the other parts of the lab. 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 outlined after the PCR procedure.
| Component | amt. in a 50 μl reaction |
Final Conc. |
|---|---|---|
| Water | add ? to achieve total of 50 μl |
_ |
| 2x Phusion Master Mix | 10 μl | 1x |
| 27F primer | ? | 0.5 μMolar |
| 1492R primer | ? | 0.5 μMolar |
| template DNA | 1 μl | __ |
| DMSO (optional) | (1.5) | 3% |
The recommendation for final primer concentration is 0.5 μM, but it can
be varied in a range of 0.2-1.0 μM if needed.
Addition of DMSO is recommended for GC-rich amplicons. DMSO is not
recommended for amplicons with very low GC% or amplicons that are
>20kb.
Thermal Cycler Program:
3 step program
| Cycle Step | Temperature | Time | # of Cycles |
|---|---|---|---|
| Initial Denaturation | 98C | 30 sec. | 1 |
| Denaturation Annealing Extension |
98C ?C 72C |
5-10 sec 10-30 sec 15-30 sec/1kb |
17-21 |
| Final Extension | 72C 4C |
5-10 min Hold |
1 |
Part C: Clean Up of pcr product using QiAquick PCR Purification Microcentrifuge Protocol or the Epoch BIoLabs GenCatch PCR CleanUp Kit
Notes before Starting:
Make sure ethanol (96-100%) has been added to Buffer PE before 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.
Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 μl pH indicator I to
30 ml Buffer PB or add 600 μl pH indicator I to 150 ml Buffer PB). The yellow color
of Buffer PB with pH indicator I indicates a pH of 7.5.
Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer
aliquots.
Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary
to remove mineral oil or kerosene.
For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).
2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is
yellow.
If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH
5.0, and mix. The color of the mixture will turn to yellow.
3. Place a QIAquick spin column in a provided 2 ml collection tube.
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 sec.
5. Discard flow-through. Place the QIAquick column back into the same tube.
Collection tubes are re-used to reduce plastic waste.
6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 sec.
7. Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless
the flow-through is discarded before this additional centrifugation.
8. Place each QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water
(pH 7.0–8.5) to the center of each QIAquick membrane, and centrifuge the
columns for 1 min at 17,900 x g (13,000 rpm). Alternatively, for increased DNA
concentration, add 30 μl elution buffer to the center of each QIAquick membrane,
let the columns stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick
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. When using water, make sure that the pH value is within this
range, and store DNA at –20°C as DNA may degrade in the absence of a buffering
agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0),
but the EDTA may inhibit subsequent enzymatic reactions.
To see if you successfully amplified DNA you can either do an electrophoresis of 1 microliter of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain (run a gel) or you can take your pcr product over to the Nanodroper and apply a 1 microliter sample and see if you have a much higher concentration of DNA than you started with. Your instructor will decide if we have time to run a gel or if you will just get a rough estimate of DNA amplification from the nanodroper.
If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time.
Give your cleaned up pcr product to your instructor to freeze, making sure it is properly labeled with your intials, lab section, soil identifier,date, etc. 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.
Identification of Cultured Bacteria from a Soil Habitat
Remember that you are working on two aspects of our Soil Diversity Project simultaneously : we are getting a sense of the total bacterial community through sequencing of the 16S rDNA from the genomic DNA isolation of our soil sample and we are also using traditional culture techniques AND DNA sequencing to isolate and identify a few of the interesting culturable bacteria. You began the isolation and selection of some culturable bacteria of specific groups from your soil sample through the use of selective and enrichment media in Lab 2. You also started a standard plate count of the culturable organisms. Today you will complete the plate count to get one kind of enumeration of the microbes in your soil community. You will also continue the process of isolating and selection of specific groups of microbes based upon unique or distinctive metabolic, morphologic, or behavioral characteristics.
Part D: Finishing the Standard Plate Count of Culturable Soil Microbial Flora & Observing Colony Morphology in the Streak Plates
Activity D-1: Find a plate that contains 30-300 colonies (there should be only one if you did your 10 fold serial dilution correctly). Count all the surface and subsurface colonies on that plate using the Quebec colony counter (as directed by your instructor). If you divide the number of colonies by the dilution factor of that plate, you will obtain the number of cultivatable bacteria per gram of soil. If it is clear that a culture plate has well over 300 colonies or under 30, designate it as "invalid" in your lab notebook.
Calculate the number of cells in 1ml of the original soil extract for at least 1 dilution. Remember that each colony represents one cell in that plated bacterial dilution.
Things to Consider:
Why did we perform so many dilutions?
Why did we plate 1ml and 0.1 ml samples from the serial dilutions tubes?
Why should you only have one plate with 30-300 colonies?
Activity D-2: Observe your streak plates. Is there a great deal of growth in zone 1 of your streak plates? How good was your technique at streaking for isolation? Did you get well isolated colonies by the 3rd or 4th section? If not, consult with your instructor and ask her to watch your technique.
Activity D-3: Observe the success of your selection/enrichment. Do your enrichment cultures show colonies that look morphologically different from those growing on the dilute nutrient agar plates? Record general observations in your lab notebook about the number and variety of colonies or the growth observed. Draw and/or take pictures of some of the most numerous and some of the most interesting colonies so you can compare their appearance this week and in the future.
Observing different colony types, based on colony size, shape, elevation, color, and consistency.
Colonial characteristics are best observed where individual colonies are well isolated. Scan your plates to find the different categories described below. Note differences in numbers and types of colonies in different media. We wish to avoid slecting any fungal colonies which generally appear as "fuzzy" growth.
Colony descriptions:
Size - pinpoint, small, medium, large, very large, and spreading.
Color - lemon yellow, golden, creamy, gray and translucent, white and opaque, red, pink and so on.
Appearance - shiny or dull
Shape (Fig 2A)- round, irregular, and so forth.
Elevation (Fig. 2A) - flattened, heaped up, concave
Consistency - dry and friable, easily broken up, sticky and stringy, and so on.
Odors - try to describe them.
Activity D-4: Each student will select 4 different well isolated colonies from any of your plates. Please streak for isolation on new plates of the same media where each colony was found AND on a medium suggested as selective/enrichment for a suspected genera. For example, oatmeal agar for suspected Streptomycetes.
Things to Consider When Selecting Media"
- What are the desired group's growth restrictions or preferences (nutritional needs, concentration of oxygen, light, pH, temperature)?
- What are the best or unique sources of C,N,S, P for this group?
If you have time today, perform a Simple staining technique or Gram Stain on these isolates and use the microscope Microscopy: Care and Use of the Compound Brightfield Microscope to examine the slide for uniformity or you may wait until you are sure you have a pure culture.
CULTURES: (Primary plates) observe-count, describe, select soil microbes for further culturing (compare anaerobic, aerobic, microaerophillic) - search web for pics of microbes.
techniques: colony morphology, number (see microbial safari= wagner) and problem solve to "discover" 2 isolation methods (serial dilution and pour plates vs streaking for isolation)
Isolation to pure colony step: #1: How will they pick what/where on plate to isolate? (secondary plates) Each student picks different 4-6 colonies and restreaks on same media (4-6 different plates). Incubate room temp.grow 24 hours, move to CR.
Microscopy introduced: select a well isolated colony and prepare a bacteria smear slide with these unknown bacteria and known Gram neg and Gram pos control bacteria in three different sections of the same slide. Gram stain the smear.
Control stocks for: gram pos, gram neg and capsule, acid fast and endospore
Simple stain Gram stain
What do you learn from this?
Links to Labs
Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12
