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.

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 25-35 cycles will require less than an hour rather than over 3.

Protocol for PCR
Add these ingredients in this order:

Component	amt. in a 50 μl reaction	Final Conc.
Water	add ? to achieve total of 50 μl	_
2x Phusion Master Mix	10	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

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?