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5.3 T7 phage selection method test
From 2013.igem.org
5.3 T7 Phage Selection Method Test
I) Purpose
- Test the size of plaques formed from x6 and x8 top agar
II) Expected outcome
- As top agar increase in concentration, the number of plaques/plate should decrease and individual plaques should become smaller.
III) Reagent record
- T7 + phage: -5 dilution from 4.26; 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 Thursday at 2:00pm using 4.6 streak; ×8 top agar stock: prepared by us on 4.3
IV) Actual procedures / observations
- i) Six test tubes were labeled control, ×6, ×6, ×8, ×8, x8, and ×8. To each of the test tubes, 0.5mL of E coli overnight liquid culture was added.
- ii) To each 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 ×6, and ×8 concentration.
- 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, ×8 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
V) Results
- The x8 plates had smaller and few plaques than the x6 plates
- The x8 plaques are about 1mm in diameter
