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5.15 Titer Test on 5.3 T7 new Phage Stock
I) Purpose
- Determine the concentration of the 5.3 T7 new Phage Stock to give an estimate for starting mutagenesis
II) Expected Outcome
- As the dilution series increases, the number of plaques on the plates should decrease
III) Reagants Used
- E5.3 T7 new phage stock; LB; BL21 E. coli from 5.14 overnight
IV) Actual Procedure
- Performed dilution series with 5.3 T7 stock; went down to -8
- Add 0.5 mL of BL21 to 4 test tubes, labelled 5, 6, 7, 8
- Add 20 uL of T7 new to each test tube. Phage concentration should correspond to the number on the test tube. Incubate at room temp for 20 minutes.
- In a 50mL centrifuge tube, combine 10mL of LB with 10mL of x2 top agar.
- Add 5mL of the combined solution to each of the four test tubes. Plate each tube and incubate at 37 C for 24 hours
V) Results
- Lots of contamination - from the plates -> this batch of LB plates have been completely disposed of.
- -8 plate had 7 plaques: This gives a phage concentration estimate of approximately 7E8 particle/20uL
- -5, -6, and -7 had overlapping plaques
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