Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period3/Exp/4.26 T7 Phage Selection Test


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Small Phage March - April Notebook: Experiments

Small Phage

4.26 T7 Phage Selection Test

I) Purpose

Test the possibility of selecting for smaller phage by increasing agar concentration

II) Expected Outcome

As top agar increase in concentration, the number of plaques/plate should decrease and individual plaques should become smaller.

III) Reagants Used

T7 + phage: fresh dilution from 4.8 -4; E coli BL21; Agar: ×2 prepared by Jordan in 500mL glass bottle; LB: prepared by us on 4.3; overnight bacteria culture: set up on Friday at 6:00pm using 4.6 streak; ×8 top agar stock: prepared by us on 4.3

IV) Actual Procedure

1. Made fresh -5 stock phage ×2

i) 180 μL LB was added to a 1.5mL centrifuge tube. 20 μL of phage stock -4 was then added.

2. Performed selection method test

i) Six test tubes were labeled control, ×1, ×2, ×4, ×6, and ×8 respectively. To each of the test tubes, 0.5mL of E coli overnight liquid culture was added.
ii) To ×1, ×2, ×4, ×6, and ×8 test tube 20 μL of -5 phage solution was added. This mixture of E coli and phage was left sitting for 20 minutes before proceeding.
iii) In a separate set of test tubes, top agar was diluted to give ×1, ×2, ×4, ×6, and ×8 concentration. For specifics, please refer to the chart in the Results section.
iv) 5mL of top agar was added to each test tube so that the concentration of the agar added matches the labels on test tube. For the control tube, ×1 agar was added. The contents of these test tubes were then plated on LB plates.
Note: ×6 and ×8 solidified as soon as the content hit the plate. As a result, top agar wasn’t spread evenly.
v) After the top agar has solidified, the plates were incubated up side down (4.26 6:30pm – 4.27 2:00pm for 19.5hr)

V) Results

1. Plate

Plate results
Plate Content Agar Results
Control 0.5mL E coli 2.5 mL ×2 agar + 2.5 mL LB Uniform lawn of E coli, with a few pinprick size clearage that might be due to air bubbles. No contamination.
×1 0.5mL E coli + 20 μL of -5 phage 2.5 mL ×2 agar + 2.5 mL LB Similar results to that seen in 4.25 T7 phage viability test
×2 0.5mL E coli + 20 μL of -5 phage 2 mL ×8 agar + 8 mL LB Smaller and fewer plaques as compared to ×1
×4 0.5mL E coli + 20 μL of -5 phage 4 mL ×8 agar + 4 mL LB Smaller and fewer plaques as compared to ×2
×6 0.5mL E coli + 20 μL of -5 phage 6 mL ×8 agar + 2 mL LB Smaller and fewer plaques as compared to ×4. Seems like the right concentration for selection.
×8 0.5mL E coli + 20 μL of -5 phage Stock No E coli growth or phage plaque seen. Possibly due to boiling agar killing off everything.

2. Selection test result


VI) Conclusion

The results of this experiment are similar to what we expected. This indicates that our selection method is working. It also seems that ×6 agar is a good place to start our selection

VII) Proposed next step

Learn the procedures to purifying phage
Start working on liquid culture to prepare for mutagen treatment.