M465:tRFLP

Part A: PCR Amplification of 16s rRNA genes with fluorescently labeled primers

The key to tRFLP is that there is variation in the length of restriction digest products of the 16s rRNA gene. Specifically, the terminal fragment (on the forward end) shows great promise of being able to distinguish many bacterial species. Using this characteristic of the 16s rRNA gene, we can 'visualize' community wide variation as a 'fingerprint' based on terminal fragment size. This is just one of many fingerprinting technique, but has been shown to be a powerful approach. (See: Terence L Marsh, Terminal restriction fragment length polymorphism (T-RFLP): An emerging method for characterizing diversity among homologous populations of amplification products, Current Opinion in Microbiology, Volume 2, Issue 3, June 1999, Pages 323-327, ISSN 1369-5274, 10.1016/S1369-5274(99)80056-3. (http://www.sciencedirect.com/science/article/pii/S1369527499800563))

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
Obain 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 sample. Do not use tape.

If your DNA 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 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 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 other liquid ingredents, 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	1-4 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	20
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 cloning next week are found later in this lab description. You will also need to set up a gel to assess the purity of your pcr product and the success of your amplification.