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4/2/13
- Created 5 liquid cultures of W3110
- 4.2 W3110 Liquid Culture
- 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
- Today we will be setting up an overnight culture of E. coli, to grow for use with phage amplification.
- We will take a 500 flask; with 50 mL LB broth, and place a culture from the W3310 E.coli culture plate to grow overnight in the 37 C incubator
- We will create a glycerol stock of the E.coli culture to save in the -80 C freezer.
4/4/13
- We made glycerol stocks W3110 E. Coli
- 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
- We plated together .5mL of BL21 with 1XLBTA.
- We separated the plates into different segments and then spot tested the following phages.
- 14 T3
- 13 T5
- 10 T2
- 20OX171
- 40ØX174
- The plates were then put in a 37°C incubator
-Results:
- There were no plaques on any of the plates.
- There was no contamination in the control quadrant.
- A uniform bacterial lawn formed with no phage infection.
4/8/13
-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
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