Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period3/Dailylog

Phage Purification

March-April

May-June

July-August

September-October

Created a 50 mL liquid culture of W3110

4.2 W3110 50mL Liquid Culture

-We also put 500 µL of 40% glycerol into cryovials for use tomorrow to prepare freezer sotcks.

4/3/13

Created 5 liquid cultures of W3110 to create 1 mL glcerol stocks of bacteria.

4.3 W3110 Liquid Culture

4/4/13

We made glycerol stocks W3110 E. Coli

4.4 Glycerol Stocks

We put .5mL glycerol in cryovials and .5mL liquid culture from the overnights prepared yesterday.

We vortexed each tube for 10 seconds and then we stored the tubes in the -80°C freezer in Dr. Grose’s lab.

4/5/13

We did spot tests of phage on E. Coli BL21

4.5 Phage Spot Test

4/8/13

-We performed a phage titer test to determine which bacteria would be most viable for our phage propagation.

4.8 Bacteria Determining Titer

-We performed a phage titer test to determine which bacteria would be most viable for our phage propagation.

We used BL21 and W3110 strains of E. Coli.

We placed 100 µL of broth into 5 ependorf tubes.

We used as phage 10 µL of 1L, 10L, 40T4, T4DOS, and T4 infected phage and placed them each in one of tubes labeled -1.

We performed a dilution series taking out 10 µL each time and placing it into the next tube, 5 times. The total volume in the last tube was 110 µL.

We added 4.5 mL of 1X top agar to .5 mL of broth and plated it on LB plates.

We spotted each concentration on the plates and incubated it overnight at 37°C.

-Results from 04/08:

Phage 10L w/ w3110 had large scale infection every concentration

Phage 10L w/ BL21 had infection in very large concentrations

Phage 1L w/ w3110 had infection in very large concentrations

Phage 1L w/ BL21 had infection in very large concentrations

Phage T4 DOS w/ w3110 had infection in very large concentrations

Phage T4 DOS w/ BL21 had infection in very large concentrations

Phage 40 T4 w/ w311o had no phage infection

Phage 40 T4 w/ BL21 had large scale infection at every concentration

Phage T4 inf w/ w311o had large scale infection at every concentration

Phage T4 inf w/ BL21 had large scale infection at every concentration

We had a significant amount of running that occurred on almost every plate, so the results were a little difficult to read. We concluded that the phage is a high enough concentration that we would have to do another phage titer to a smaller dilution in order to determine actual concentration. The controls were all a lawn of bacteria.

4/10/13

-We performed titers of T4 infected phage from off of two separate E. Coli strands, BL21 and W3110.

We added .1mL of the liquid culture to 1 mL of both strands of bacteria.

After allowing infection to occur overnight, we centrifuged the tubes to separate the bacteria from the phage.

We separated the supernatant into a new eppendorf tube, and used 10 µL as the 0 concentration.

We then performed a phage titer using five tubes of 90 µL LB each, adding 10 µL from each one to the next.

Using a micropipette we spotted 2µL of each dilution onto both 4.5 ml 1x plates and .5 mL W3110 plates.

To avoid smearing, we allowed the plates to sit for over an hour and then we incubated them at 37 C overnight.

-Results:

The concentrations we used are still way too strong. We will have to dilute the concentration even further if we want to see anything more than cleared plaques.

4/12/13

-We watched presentations from the Cholera Group Today