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BISC314: Environmental Microbiology

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LAB #8: Identifying your Isolates by PCR and Sequencing

The organisms you've been working with for your cheese and yogurt exhibit interesting properties. At this point, you should know what they look like, what their gram stain is, whether or not they interact with each other via growth inhibition or promotion, if they produce AI detectable by CV026, if they harbor phage. Today we will use a molecular tool to identify what genus your organism is from. We will be amplifying the 16S rRNA gene from the frozen aliquots you prepared in lab 2. This gene is well conserved among all cellular life forms - so much so that you can build a phylogenetic tree of all cellular life using an alignment of the sequences from this gene (or its homolog). To see and learn more about this tree read the primary paper by Carl Woese here.

Boiling lysis of our isolates

1. Take your 1.5 ml tubes containing your isolates in water out of the freezer and place on ice.
2. Cap each of the tubes securely with a cap lock.
3. Place them in a floating rack and add them to the boiling water. Set a timer for 5 minutes.
4. After the time is done, take your rack out of the water and place on ice immediately.
This is the lysate we will use for PCR below

We are utilizing "universal" primers to amplify the 16S rRNA gene from the isolates. Why are these primers called "universal"? Can you think of how they were designed? What organisms may they detect? Which may be excluded?

For each PCR reaction you will need to generate the following mix:

Reagent Quantity Calculated Total
Phusion 2x Mix 10 ul
10uM F primer 0.5 ul
10uM R primer 0.5 ul
Water 8 ul
lysate 1 ul ----

You'll be doing one PCR reaction for each of your isolates. So, for example, if I had 10 isolates, I would make up what's called a master mix. I'd need 11 total reaction (one for a "blank" that won't have DNA template added). Add 1 to this number so that you end up with enough total mix. That leaves me with 12. I'd multiply each of the amounts above by 12 to get this table:

Reagent Quantity Calculated Total
Phusion 2x Mix 10 ul 120 ul
10uM F primer 0.5 ul 6 ul
10uM R primer 0.5 ul 6 ul
Water 8 ul 48
lysate 1 ul ----

Remember to keep this all on ice! and Note that I do not multiply my lysate by 12!

1. Make your master mix following the calculated amounts above.
2. Gently mix the reaction tubes by flicking - do not pipette up and down!
3. The mix is a total of 19 ul per reaction. Label your PCR tubes according to the isolate lysate that will be added. To each tube add 19 ul of master mix. If there are bubbles in your tubes, spin them down before adding your lysate.
4. Add 1 ul of your lysate to each tube.
5. Take the tubes over to the PCR machine and we'll start the reaction. It should take about 1 hour to complete.

While the PCR machine is running, we'll take a short field trip to visit the organisms you've identified in your life list.

6. After the PCR cycle is done, take your 200 ul tubes out of the machine and place them on ice.
7. We will be transferring all 20 ul of your reactions to a 96-well plate. You need to reserve a spot on that plate by signing up on the spreadsheet located in the course conference. This way you'll know what spots are taken and when your sequences come back, we'll be able to give you your own sequences and not your classmates'.

Prof. Newton will send this plate for sequencing. Our results should come back in a few days.

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