BISC209: Lab3

Lab 3: Continue Soil Microbial Communities & Diversity Project

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 with 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.

==Finishing the Standard Plate Count of Culturable Soil Microbial Flora & Observing Colony Morphology in the Streak Plates==

In this culture identification project, your goal will be to take a look at the culturable microbial population from your standard plate count on all purpose media and, at the same time, begin to isolate, identify, and assess the role of 4 bacteria that contribute to your soil community from either these plates or the isolation streak plates.

Observe your pour plates and count all the surface and subsurface colonies on the Quebec colony counter as directed by your instructor. If it clear that a plate has well over 300 colonies, designate it as "invalid" in your lab notebook and go on to the next plate.

Enter your results in an appropriate table that you have made in your lab notebook. Select the valid count, that is one that falls between 30 and 300 colonies. Multiply this count by the dilution factor of the plate and enter the total plate count per ml of soil. If two colony counts fall between 30 and 300, this generally indicates a pipetting or other dilution error. Consult with your instructor on how to proceed.

Observe your streak plates. How was your technique at streaking for isolation. Did you get well isolated colonies on any of the dilutions? Did your enrichment media show colonies that look morphologically different from those growing on the dilute nutrient broth? Spend some time closely evaluating the number of different types of colonies that you have on your different plates. Draw some of the most numerous and some of the most interesting. Save all your plates for another week wrapped in saran wrap or inside a sealable plastic bag.

Protocol: 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 tiped 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

PCR Purification

Protocol: 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.

7. Place each QIAquick column into a clean 1.5 ml microcentrifuge tube.

8. 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. Refer to Table 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.

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.

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 something that looks isolated and stain and compare to stock cultures.

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 in the Soil Microbes Project

Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9