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 (10x=0.25 Tris, 1.9M Glycine, 13mM EDTA) with SybrSafe™ 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 (0.25 XC, 30% glycerol, 0.1mg/mlRNase) 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. These unique code names for your isolates shoul continue the scheme we used for the culture-independent bacteria. For example, all isolates from the Durant Camellia house begin with S; Tropical house isolates T begin with T and isolates from around the bamboo in the Hydrophyte house begin with H. If you are in the Tues lab, your isolates will start with 300 (odd numbers) and if you are in the Wed lab, your isolates start with 400 (even numbers). Each student will be assigned a unique group of numbers within that range to use for coding your isolates as S301 or H490, etc. Additionally, please label the gel template with the group of numbers for your isolates as, example: Tues.; Red; Student initials-S301-S306. Make a copy of your habitat team's part of the template in your lab notebook and make sure you know which code number corresponds with which isolate you have been gathering test results.
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. We will use these same code identifiers when we send off the pcr products for 16S sequencing so your pcr product microfuge tubes MUST have these code identifiers and they must be clear.
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 out all the successful amplicons for DNA sequencing of the 16s rRNA gene. We must also send an appropriate primer. What's the sequence of such an appropriate primer? Your instructor will give you more instructions on whether you or she will load your successful amplicons (pcr products)into a 96well plate for freezing and shipping to Agencourt/Beckman.
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.
1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.
2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.
3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.
4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.
5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.
6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.
7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.
8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.
9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.
10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.
11. Take off your lab coat and store it in the blue cabinet with your microscope.
12. Wash your hands.
Study for your Lab Practical
Continue to wind down and finalize the characterization of your culturable isolates.
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