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Team:BYU Provo/Notebook/Phage Purification/Springexp/Period2/Dailylog
From 2013.igem.org
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5/13/13 - Thankfully we don’t have to use the ultracentrifuge to achieve 10,000 g. We also won’t need to order any special centrifuge tubes, so we should be able to start experimentation on Wednesday. -We added 1 mL of w3110 and 1 mL of BL21 to 24 mL of LB each and placed them both into the 37C incubator. After an hour of incubation we infected the BL21 Ecoli with t7 phage and left to incubate until wednesday. We will infect the w3110 tomorrow with T4
5/14/13 - Today we came in and infected 24 mL of LB with 1 mL of w3310 and put in the 37C incubator overnight. I also infected 25 mL of w3110 with 100 µL of t4 phage and left overnight in the 37C incubator overnight.
5/15/13 - We added 2.5 ml of LB to the 24 mL of t4 infected W3110 to make a final volume of 25 mL. - We added 92.5 µL of DNAse I and 12.5 µL of RNAse A into the 25mL of t4 infected w3110 Ecoli and the t7 infected BL21. - We then added 50 µL of chloroform to each lysate and let it incubate for 30 min at room temperature. - We then added .73 g of solid NaCl to both lysates and let it sit at 4 C for 1 hour.
5/16/13 -Centrifuged at 8000 rpm (10,000 g) for 15 minutes. -Removed supernatant and added phage suspension buffer
-Left overnight
5/17/13 - Centrifuged (4C) at 5500 rpm (5,000 g) for 10 min. - Added an equal volume of chloroform, let sit for 1 min - Centrifuged (4C) at 5500 rpm (5,000g) for 15 min. - Saved the phage containing supernatant in eppendorf tubes, placed in the fridge until we decide what to do with them. -Over the weekend: come up with a community outreach program!
5/20/13 - Performed a titer on both T4 and T7 to see if purification was successful.
5/22/13 - Repeated T4 titer as the last titer showed contamination. - Created a new 25 mL stock of BL21 E. coli
5/24/13 - Ran a CsCl gradient to further purify phage from bacterial debris.
5/26/13 - Began the PEG purification.
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