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Team:BYU Provo/Notebook/SmallPhage/Springexp/Period1/Exp/5.9 T7 selection method test
From 2013.igem.org
5.9 T7 Phage Selection Method Test #2
I) Purpose
- Test the size of plaques formed from x6 and x8 top agar
- To practice working with thick 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: -7 dilution from 5.8, specifically, -7 dilution from stock 5.6; 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 Wednesday at 4:00pm using 4.6 streak; ×8 top agar stock: prepared by us on 4.3
IV) Actual procedures / observations
- i) Warmed up the plates and pipettes by placing them in the 37 C incubator for 3 hours
- ii) Ten test tubes were labeled control, ×6, ×6, x6, x6, x8, x8 ×8, ×8, x8, and ×8. To each of the test tubes, 0.5mL of E coli overnight liquid culture was added.
- iii) To each test tube 20 μL of -7 phage solution was added. This mixture of E coli and phage was left sitting for 20 minutes before proceeding.
- iv) In a separate set of test tubes, top agar was diluted to give ×6, and ×8 concentration.
- v) 5mL of top agar was added to each test tube so that the concentration of the agar added matches the labels on test tube. The contents of these test tubes were then plated on LB plates.
- vi) After the top agar has solidified, the plates were incubated up side down
V) Results (5.10)
- We were able to get much prettier plates than last time. x8 plates had fewer and smaller plaques than x6. Four plates are shown in the photo here
