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| - | <font size="4"> '''5/1/13''' </font>
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| - | -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.
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| - | :Plan to make an excel sheet that will calculate the needed volumes of reagents for the volume of lysate used.
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| - | :Plan how many days it will take to complete the procedure - plan in breaks and time
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| - | -Questions to ask Dr. Grose:
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| - | :What is less expensive/better - Freeze thaw or use lithium chloride
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| - | ::Answer: Try both
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| - | :Best method for determining phage purification
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| - | :Step 3 on 3.3.1.1
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| - | :CsCl gradient - we may need someone with experience to help
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| - | :Should we plan on using CsCl? Or just plan on PEG? (time to set up gradient)
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| - | :Step 7 on 3.2.1 (dialysis??)
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| - | <font size="4"> '''5/3/13''' </font>
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| - | -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.
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| - | <font size="4"> '''5/6/13''' </font>
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| - | -We created more bacteria stock of W1130 and BL21
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| - | :For BL21 we put 1 mL of BL21 into 24 mL of LB and incubating overnight at 37 C
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| - | :For W3110 we put 1 mL of W3110 into 24 mL of LB and incubating overnight at 37 C
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| - | :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.
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| - | -We are still waiting on supplies for propagation
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| - | -We learned that the T7 Phage requires 24-48 hours to infect.
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| - | -Results:
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| - | :Our W3310 didn’t grow in the liquid culture we will have to create a new one.
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| - | <font size="4"> '''5/8/13''' </font>
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| - | -Most of our reagents came except for our DNase. Hopefully it will come by Friday.
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| - | -We created more stock of both BL21 and W3110.
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| - | :We added 24 mL of LB to two erlenmeyer flasks.
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| - | :We added 1 mL of BL21 to one flask and 2 mL of W3310 to the other.
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| - | :The W3310 was then incubated for an hour at 30 C and the BL21 was incubated for an hour at 37 C
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| - | <font size="4"> '''4/8/13''' </font>
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| - | -We performed a phage titer test to determine which bacteria would be most viable for our phage propagation.
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| - | :We used BL21 and W3110 strains of E. Coli.
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| - | :We placed 100 µL of broth into 5 ependorf tubes.
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| - | :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.
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| - | :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.
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| - | :We added 4.5 mL of 1X top agar to .5 mL of broth and plated it on LB plates.
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| - | :We spotted each concentration on the plates and incubated it overnight at 37°C.
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| - | -Results from 04/08:
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| - | :Phage 10L w/ w3110 had large scale infection every concentration
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| - | :Phage 10L w/ BL21 had infection in very large concentrations
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| - | :Phage 1L w/ w3110 had infection in very large concentrations
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| - | :Phage 1L w/ BL21 had infection in very large concentrations
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| - | :Phage T4 DOS w/ w3110 had infection in very large concentrations
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| - | :Phage T4 DOS w/ BL21 had infection in very large concentrations
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| - | :Phage 40 T4 w/ w311o had no phage infection
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| - | :Phage 40 T4 w/ BL21 had large scale infection at every concentration
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| - | :Phage T4 inf w/ w311o had large scale infection at every concentration
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| - | :Phage T4 inf w/ BL21 had large scale infection at every concentration
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| - | :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.
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| - | <font size="4"> '''4/10/13''' </font>
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| - | -We performed titers of T4 infected phage from off of two separate E. Coli strands, BL21 and W3110.
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| - | :We added .1mL of the liquid culture to 1 mL of both strands of bacteria.
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| - | :After allowing infection to occur overnight, we centrifuged the tubes to separate the bacteria from the phage.
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| - | :We separated the supernatant into a new eppendorf tube, and used 10 µL as the 0 concentration.
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| - | :We then performed a phage titer using five tubes of 90 µL LB each, adding 10 µL from each one to the next.
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| - | :Using a micropipette we spotted 2µL of each dilution onto both 4.5 ml 1x plates and .5 mL W3110 plates.
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| - | :To avoid smearing, we allowed the plates to sit for over an hour and then we incubated them at 37 C overnight.
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| - | -Results:
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| - | :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.
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| - | <font size="4"> '''4/12/13''' </font>
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| - | -We watched presentations from the Cholera Group Today
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