BISC314:Lab6: Difference between revisions
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Today we will be using a drug, mitomycin C, to induce production of active phage particles from lysogenized phage in our LAB's. Your overnight cultures will serve as inocula for a larger culture of your LAB isolates. We will then add mitomycin C and measure the OD600 at 20 minute intervals. How do you think the OD600 will change if a lysogenized phage becomes lytic? <br> | Today we will be using a drug, mitomycin C, to induce production of active phage particles from lysogenized phage in our LAB's. Your overnight cultures will serve as inocula for a larger culture of your LAB isolates. We will then add mitomycin C and measure the OD600 at 20 minute intervals. How do you think the OD600 will change if a lysogenized phage becomes lytic? <br> | ||
- Measure the OD600 of your overnight culture of LAB. You will do this two ways: either using the side-arm spec or by using a cuvette in the regular spec. You will use the cuvette method first, as it is more accurate for higher density cultures (like your overnight culture) but throughout your time course, you will use the side arm spec method. <br> | |||
''regular spec use'' <br> | ''regular spec use'' <br> | ||
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5. Remember - your sample is a 1:10 dilution of your original culture! Multiply the number you get from the spec by 10 to get the true OD600 / ml. <br> | 5. Remember - your sample is a 1:10 dilution of your original culture! Multiply the number you get from the spec by 10 to get the true OD600 / ml. <br> | ||
''side-arm spec use'' <br> | |||
1. The side arm spectrophotometer takes a measurement of OD600 through the side arm of the flask. This makes it easy to measure the OD600 without contaminating your culture. <br> | |||
2. Insert the culture tube in the spectrophotometer. Take note of the measurement as it appears on the instrument. <br> | |||
''Beginning the experiment: Mitomicin C treatment'' <br> | ''Beginning the experiment: Mitomicin C treatment'' <br> | ||
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1. Aseptically add the volume necessary of your O/N culture to two 10 mL culture tubes containing fresh LAB liquid media. One of these you will label as your experimental tube and the other will be your control. <br> | 1. Aseptically add the volume necessary of your O/N culture to two 10 mL culture tubes containing fresh LAB liquid media. One of these you will label as your experimental tube and the other will be your control. <br> | ||
2. Aseptically add mitomycin C to your LAB experimental culture. Note the time in your lab notebook and set a timer for 20 minutes. <br> | 2. Aseptically add mitomycin C to your LAB experimental culture. Note the time in your lab notebook and set a timer for 20 minutes. <br> | ||
3. | 3. Measure the OD600 of your 10 mL cultures every 20 minutes for 2 hours. You'll fill in the table below: <br> | ||
<table border="2"> | <table border="2"> |