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
The labeled primers are light sensitive, so it is good to keep these out of direct light while setting up your reactions and during all down stage processing
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:

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

Remember to do a negative control

Thermal Cycler Program:
3 step program

Cycle Step	Temperature	Time	# of Cycles
Initial Denaturation	94°C	2 min	1
Denaturation Annealing Extension	94°C 55°C 72°C	35 sec 45 sec 2 min	30
Final Extension	72°C 4°C	2 min Hold	1

This PCR program seems a bit long, but we use ample time to for extension to prevent any artifacts in t-RFLP profiles. Once the Rxn has completed, visualize your product on a 1% Gel to make sure you successfully formed products. If everything work, you can store you PCR products at -20°C until after spring break. At that time you will digest the PCR products with MspI to form the terminal fragment bands that will be visualized.

Part B: Restriction Digest of Amplified 16s rRNA genes

The next step in our t-RFLP pipeline will be to digest the amplified PCR products. We are going to use the restriction enzyme MspI which cleaves DNA at the palindromic sequence C*CGG. The enzyme will cut the ~1400 bp 16S rRNA gene in many locations, but by using the FAM fluorescent tag we will only be looking at the Terminal (5') end of the PCR product.

Today's digests will be done at a volume of 20 µl. In this reaction we will need ~ 200-500 ng of PCR product. Make sure you correctly quantify and calculate the vol of PCR product needed to reach this range before proceeding. The digests need to be done at 37°C for 4 hrs. I suggest using a thermocycler to make this more efficient; however, an incubator or waterbath will work in a pinch.

t-RFLP Digest Table

Component	amt. in a 20 µl reaction	Final Conc.
Purified DNAase free Water	You want to achieve a total of 20 µl rxn vol. Add based on final DNA conc.	_
10X Buffer	2 µl	1 X
BSA (10 mg/µl)	0.2 µl	100 µg/µL
MspI (10 U/µl)	0.5µl	5 U
PCR Product	x µl	optimum is 200 ng - 500 ng of DNA/reaction

Remember to do a negative control

I recommend making a Digest Master Mix so that you will have enough to set up all of your Digests. Individual Digests should be set up in 200 µl PCR tubes. Once you set these up put them at 37°C for 4 hrs to completely digest the templates. You can then place the products at -20°C until the next class time.