M465:16S rRNA gene lab work

Creating Lysates from your Isolates

One of the big advantages of the molecular revolution in microbial ecology is that we can actually find, based on 16S rRNA gene sequences, the identity of your isolates. In order to do that, we will follow the protocol below which first involves liberating the DNA in your isolate, then amplifying the 16S rRNA gene, then sequencing it. The bioinformatics analysis of this sequence will be covered next time we meet.

Isolate Genomic DNA From Your Sample
Please wear gloves during this protocol

1. For each of your isolates, label a 200 ul PCR tube and fill it with 100 ul of water.
2. Using a sterile loop, transfer a small bit (barely visible) of your isolate into the 100 ul solution in the PCR tube - make sure to shake the isolate off the loop so that it is in the water
3. Cap and vortex the PCR tube for 10 seconds.
4. Place in the PCR machine at 95C for 10 minutes.

Storing DNA:
Because you've just made a lysate of your isolate, remember that this solution contains all cellular contents, including DNAses -- enzymes that break down that very DNA we are trying to amplify. You can store your lysates at -20C in the freezer in the lab but make sure to create new ones if you freeze-thaw them more than once.

We will next attempt to amplify only bacterial rRNA genes 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 exponential amplification of the 16s rRNA gene from the template DNA. After 30 cycles of polymerase chain reaction in a thermal cycler, the result will be a pcr product containing hundreds, if not thousands, of the 16s rRNA gene.

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:
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 a extremophilic bacterium, Thermus aquaticus, that lives and reproduces in boiling hot springs. We are not using Taq for our pcr but a different polymerase, Finnzyme's Phusion High-Fidelity Polymerase, a proprietary reagent that uses a novel heat-stable Pyrococcus-like enzyme. Phusion DNA Polymerase generates long templates with a 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
Obtain a tiny 0.2ml pcr tube from your instructor. 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 isolate. Do not use tape.

you will add 4 μL of lysate to your PCR tube with master mix. 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 the P10 or P20 pipettes. Look at the tip after you draw up your measured volume to make sure you have liquid there.

Dispense the template DNA into the liquid directly, watching to make sure that the liquid has left the pipette tip.

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; they will show you where the thermal cycler is located in JH 022. Your instructor will start the reaction when everyone's tubes are loaded.

Component TABLE

Component	amt. in a 20 μl reaction	Final Conc.
Purified DNAase free Water	4 μL already in tube. Want to achieve total of 20 μl reaction vol. Add from 0 - 3μl	_
2x Phusion Master Mix	10 μl	1x
27F primer	1	0.5 μMolar
1492R primer	1	0.5 μMolar
template DNA	4 μl	optimum is 100ng of DNA/reaction

The cycling program is shown below.

Thermal Cycler Program:
3 step program

Cycle Step	Temperature	Time	# of Cycles
Initial Denaturation	98C	5 min.	1
Denaturation Annealing Extension	98C 55C 72C	10 sec 30 sec 30 sec	30
Final Extension	72C 4C	10 min Hold	1

While the 16S rRNA genes from all of the bacterial species in your DNA are being amplified in the thermal cycler, you will have about an hour to work on any other parts of your project.
After the PCR reactions are complete, 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 sequencing next time. You will also need to set up a gel to assess the purity of your pcr product and the success of your amplification.

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 a small fraction of the volume 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 on OnCourse 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?

Protocol for making a 1% agarose gel

In order to run out your PCR products you will first pour a 1% agarose gel. The recipe for the gel is 1.0% agarose (w/v) in 1x TBE buffer (10x=890mM Tris, 890mM Boric Acid, 20mM EDTA) with SybrSafe™ stain. Your instructor will add the SybrSafe stain to your molten agarose after you prepare it. Before you start, prepare your agarose gel, comb, and reservoir as you are instructed

Protocol: 1. Using a graduated cylinder, fill a glass, 250 mL flask with 100 mL of 1x TBE buffer.
2. Using the top loading balance, measure out 1 gram of Agarose
3. Pour the agarose into the flask containing the TBE buffer and stir to mix. The agarose won't go into solution here but needs to be heated
4. Place the flask inside the microwave and heat for 1 minute, maximum power. At the end of the minute, swirl the solution to mix and place back in the microwave.
5. Heat for 1 minute again, this time watching closely to make sure it does not boil. At the end, swirl to mix and bring to your instructor for the sybersafe dye
6. After your instructor adds the SyberSafe, pour your gel into the mould. It will take ~ 25 minutes to set.

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
1. Load 5 microliters of your PCR product on your gel by placing the 5 microliters of your pcr product as a spot on a small piece of parafilm and adding 5 microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the loading dye by pipetting up and down on the spot, on the parafilm, before loading all 10 microliters into a lane of the 1% agarose gel.
2. Record which lane is loaded with which product. Be sure to leave the first two lanes and the last lane empty for the 1kbp ladder, the positive control and the negative water control.

• • Note that 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 TBE buffer is used in this electrophoretic separation (89mM Tris, 89mM Boric acid, 2.0mM EDTA. The gel will be run at 120V for approximately 30 minutes.
    
    

Part C: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit

Before we can ligate our bacterial 16s rRNA genes into vector plasmids, we must remove interfering dNPTs, primers, and other small degraded DNA. We will use a column that separates DNA by size. Since the reagents and column materials in the kit we will use are proprietary, we won't know exactly what is going on at each step but, basicially, we will apply our pcr product to a column of a particular density, wash away elements too small to be trapped in it, and elute off the larger fragments of DNA (that should be ~1500bps if our pcr amplification of the 16s rRNA genes in our soil genomic DNA was successful). Notes before Starting:
95% ethanol has been added to Buffer WS before first time use (see bottle label for volume).
All centrifuge steps are carried out at 17,900rfc (~13,000 rpm in a microcentrifuge) in a conventional tabletop microcentrifuge at room temperature.

Procedure
1. Measure 500 μl of Buffer PX using your P1000 and add part of it to your 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 all of the pcr product/bufferPX mixture created in step 1 (up to a maximum of 700μL total volume) 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 applied all the pcr product to the spin column in step 3, skip this step and proceed to step 6. 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.

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 spin column by applying 700 μL of Buffer WS. Note that WS Buffer is different than the buffer used in step 6!!! Centrifuge the column for an additional 1 min. Check that ALL the buffer is in the flow-through, if there is buffer remaining in the spin-column, re-spin if needed. Discard the flow-through.

9. Centrifuge the spin-column in the same collection tube at full speed for 3 more minutes to remove ALL 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 50μl 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 in order to determine the success of this amplification and clean-up.

IMPORTANT NOTES for using this kit: Ensure that the elution buffer (EB) 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.
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 agent.

Make sure your pcr product is clearly labeled. Your instructor will measure the new DNA conc. using the nanodrop and post those concentrations for you in ng/μL. In the next lab you will use the most successful of your team's pcr amplifications of 16s rDNA and use those pcr products to ligate the 16s rRNA gene genes from your bacteria community into a special genetically engineered cloning vector. Once the a bacterium's 16s rRNA gene is incorporated into those vector plasmids, we will transform competent genetically engineered E. coli bacteria with a plasmid. Transformants (bacteria that have taken up a vector plasmid and express its genes) will be plated on selective media to find cells containing the 16s rRNA gene insert. Eventually we will send away some of those E. coli for sequence analysis to determine the identity of some of the bacterial community members in your original soil sample.