LAB 7: ID of culturable soil bacteria: Agarose Gel Electrophoresis & Clean-UP of PCR Amplified 16S rDNA from pure colonies of soil bacteria
Agarose Gel Electrophoresis
You will run your cleaned-up pcr products on a gel of 1.0 to 1.2% SeakemLE agarose solution (w/v) in 0.5x TBE buffer with Sybr Green stain to assess your success at amplifying 16s rDNA from each of your soil bacteria that you want to identify.
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.
RUNNING THE GEL
Keep your pcr products on ice until your instructor tells you that the gel is ready to load.
1. Pipet 1μL of loading dye for each of your pcr products on a piece of parafilm (use a P10 and space out the drops so they are not near each other). You should have 5 pcr products per person: 4 bacteria to id and one neg (water) control.
2. Using a new tip, pipet 5μL of a pcr product into one of the drops of loading dye, mix with the pipet tip and then draw up all of it and dispense it into a well of the gel.
3. Fill out the gel template which identifies which bacterial strain is in each well.(Use a unique code name such as TR-1 --Tues.lab Red group- strain 1.) Make a copy of the template in your lab notebook.
4. Your instructor will add the ladder marker and a positive control and start the current when all students have loaded their samples.
5. When the gel has finished (usually 45 min. at 100volts), your instructor will photograph it under UV light, label the lanes from the template, and post the photo to the data folder on the conference.
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 clean up all the successful amplifications.
PCR CLEAN UP with EXOSAPit
We will have to clean up our successfully amplified pcr products to remove any unconsumed dNTPs and primers remaining in the PCR product mixture that will interfere with sequencing. We will use a kit. ExoSAP-IT contains two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, together in a proprietary buffer. 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.
ExoSap master mix/per reaction= ExoSapIt enzymes 0.25μL with 3.25μL sterile water
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 template sheet. 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 sending away for sequencing. When the clean up is complete, transfer 3 microliters of each pcr product in to a well of a 96 well plate. Carefully fill out the template so we know in which wells your samples are found and that your code names for your samples are unique. We should get our sequencing data back within 2 weeks.
Culturable Bacteria Identification continued
Read last weeks tests; Continue with new differential ID tests; Start
Antibiotic sensitivity and nitogen recycling tests;
add other "role" tests as needed. Use the directions in the Protocols section of the wiki.
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