M465:tRFLP: Difference between revisions
Line 20: | Line 20: | ||
{| border="1" | {| border="1" | ||
|+ | |+ | ||
! Component !! amt. in a | ! Component !! amt. in a 50 μl<br>reaction !! Final Conc. | ||
|- | |- | ||
! Purified<BR>DNAase free <BR> Water | ! Purified<BR>DNAase free <BR> Water |
Revision as of 16:16, 5 March 2013
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
Component TABLE
Component | amt. in a 50 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.