Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/CsClGradientPhagePurification

Overview

March-April

May-June

July-August

September-October

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