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

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: [[Team:BYU_Provo/Phage_Purification|Overview]]
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: <u> '''Phage Purification''' </u> </font>
: [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
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<html> <a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period3/Dailylog"><< Previous</a>
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<a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Springexp/Period1/Dailylog" style="display:block;float:right;">Next >></a>
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<font size="4"> '''4/15/13''' </font>
<font size="4"> '''4/15/13''' </font>
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<font size="4"> '''5/1/13''' </font>
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<font size="4"> '''04/16/13 - 04/30/13''' </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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- Spring Break
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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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<a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Springexp/Period1/Dailylog" style="display:block;float:right;">Next >></a>
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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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Latest revision as of 00:02, 28 September 2013


Phage Purification March - April Notebook: April 15 - April 30 Daily Log



Phage Purification
March-April
May-June
July-August
September-October



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4/15/13

-We presented our project today. -There were a few things that we needed to do better.

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.


04/16/13 - 04/30/13

- Spring Break

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