M465:tRFLP: Difference between revisions
Line 17: | Line 17: | ||
'''We want to use 10-20 ng of template in each 50 µl PCR reaction'''<BR><BR> | '''We want to use 10-20 ng of template in each 50 µl PCR reaction'''<BR><BR> | ||
Set up your PCR reactions according to the following table <BR> | Set up your PCR reactions according to the following table: <BR> | ||
'''Component TABLE '''<BR> | '''Component TABLE '''<BR> | ||
Line 58: | Line 57: | ||
|- | |- | ||
|} | |} | ||
'''Remember to do a negative control''' | |||
The cycling program is shown below. <BR><BR> | The cycling program is shown below. <BR><BR> | ||
Line 70: | Line 69: | ||
|- | |- | ||
! Initial Denaturation | ! Initial Denaturation | ||
| | | 94°C | ||
| | | 2 min | ||
| 1 | | 1 | ||
|- | |- | ||
! Denaturation <br>Annealing<br>Extension | ! Denaturation<br>Annealing<br>Extension | ||
| | | 94°C <br> 55°C<br> 72°C | ||
| | | 35 sec <br>45 sec <br> 2 min | ||
| | | 30 | ||
|- | |- | ||
! Final Extension | ! Final Extension | ||
| | | 72°C <Br> 4°C | ||
| | | 2 min <BR> Hold | ||
| 1 | | 1 | ||
|- | |- |
Revision as of 16:31, 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
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
The cycling program is shown below.
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 |
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.