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 front 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 variation. 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))

Protocol for t-RFLP PCR
All reagents and PCR tubes can be obtained from your instructor
Label tubes with a fine tipped Sharpie on the top and side with the code name for your sample. Do not use tape.

We are going to set up a 50 µl PCR reaction for each of our samples
We want to use 10-20 ng of template in each 50 µl PCR reaction

Set up your PCR reactions according to the following table
Remember to do a negative control

Component TABLE

Component	amt. in a 50 µl reaction	Final Conc.
Purified DNAase free Water	You want to achieve a total of 50 µl rxn vol. Add based on final DNA conc.	_
25 mM IT Buffer	5 µl	2.5 mM
dNTPs (10 mM each)	1.25 µl	250 nM each
BSA (10 mg/µl)	1.25 µl	250 µg/mL
8F FAM Primer (10 µM)	1 µl	250 nM
1492R Primer (10 µM)	1 µl	250 nM
GoTaq (5 U/µl)	0.5 µl	2.5 U
template DNA	x µl	optimum is 10ng - 20 ng 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.