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 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.
Culturable Bacteria Characterization continued
Continue following the protocols for Tests to determine the role of a soil isolate
Read and assess tests.
Continue with/add new differential ID test
If you have an interesting new isolate it is not too late to check its Gram stain reaction and morphology, motility, and a few simple "role" tests performed over the last few weeks. Select tests that can be completed within 2 more weeks. By this point, you should be wrapping up collecting test results.
Study for your Lab Practical
Continue to wind down and finalize the characterization of your culturable isolates.
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