Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period4/Dailylog

Overview

March-April

May-June

July-August

September-October

For one, we need to present better background and show how it ties into our research, specifically the need for a capsid library.

I feel like we could have been a bit more engaging and better rehearsed as well.

We will need to find a way to test the capsids for viability, and somehow characterize them.

5/1/13

-Today we are going to create a plan to follow throughout the semester. We also talked with Jordan about reagents for performing phage purification on Monday. We also reviewed the methods for our procedure.

Plan to make an excel sheet that will calculate the needed volumes of reagents for the volume of lysate used.

Plan how many days it will take to complete the procedure - plan in breaks and time

-Questions to ask Dr. Grose:

What is less expensive/better - Freeze thaw or use lithium chloride

Answer: Try both

Best method for determining phage purification

Step 3 on 3.3.1.1

CsCl gradient - we may need someone with experience to help

Should we plan on using CsCl? Or just plan on PEG? (time to set up gradient)

Step 7 on 3.2.1 (dialysis??)

5/3/13

-We created an excel sheet to calculate the amounts of each reagent for the experiment we will be running on Monday.  :This will allow for easy calculations each time we run the experiment.

5/6/13

-We created more bacteria stock of W1130 and BL21

For BL21 we put 1 mL of BL21 into 24 mL of LB and incubating overnight at 37 C

For W3110 we put 1 mL of W3110 into 24 mL of LB and incubating overnight at 37 C

We also centrifuged 5 mL of t7 lysate in 5 1 mL ependorf tubes at 3050 rpms and separated the phage supernatant into 5 1 mL ependorf tubes and stored it in the fridge.

-We are still waiting on supplies for propagation

-We learned that the T7 Phage requires 24-48 hours to infect.

-Results:

Our W3310 didn’t grow in the liquid culture we will have to create a new one.

5/8/13

-Most of our reagents came except for our DNase. Hopefully it will come by Friday. -We created more stock of both BL21 and W3110.

We added 24 mL of LB to two erlenmeyer flasks.

We added 1 mL of BL21 to one flask and 2 mL of W3310 to the other.

The W3310 was then incubated for an hour at 30 C and the BL21 was incubated for an hour at 37 C

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