5.3 T7 phage selection method test

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Revision as of 18:22, 21 May 2013

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