PCR CLEAN UP with EXOSAPit
We will have to clean up our reactions for sequencing to remove the excess dNTPS, primers, and pcr product contaminants using EXOSAP it.
When PCR amplification is complete, any unconsumed dNTPs and primers remaining in the PCR product mixture will interfere with these methods. ExoSAP-IT removes these contaminants.
ExoSAP-IT contains two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, together in a proprietary buffer. It removes unwanted dNTPs and primers from PCR products. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. Shrimp Alkaline Phosphatase removes the remaining dNTPs from the PCR mixture.<BR
PROTOCOL for EXOSAPIT
For those pcr reactions that resulted in a single product of the expected size, combine 1.5 microliters of your pcr reaction with 3.5 microliters of EXOSAPIT master mix in clearly labeled pcr tube of your team color. Place those tubes in the thermal cycler and record the position of each of your samples on the 96 well sheet provided. For any amplifications that were not successful or resulted in multiple fragments of the wrong size, consult with your instructor about whether or not to include them.
The PCR program will be: 37C for 30 min. and 80C for 15 min. (to denature the enzymes). We will use 3 microliters of the ExoSapit reaction for sequencing.
Agarose Gel Electrophoresis of PCR products
DNA is uniformly negatively charged and will,therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA.
You will run your cleaned-up pcr products on a 1.5% agarose gel to assess your success at amplifying 16s rDNA from each of your soil bacteria that you want to identify. How will you judge a successful amplification? How many fragments and of what size do you expect to see? Remember that you used the same "universal" bacteria primer pairs that we used in our other amplification of 16s rDNA from the genomic soil DNA extract but you used a less "picky" DNA polymerase, Taq, rather than an expensive proof-reading polymerase.
We will send all the pcr products from all successful amplifications away for sequencing of the 16s rDNA. Your instructor will explain how to prepare them for sending away.
Culturable Bacteria Identification continued
Read last weeks tests, Continue with new tests
Enterotube? Antibiotic sensitivity etc
set up nitogen recycling tests
add other "role" tests
Links to Labs