Week 3

6/25
Today I counted and split my 293 FT cells, putting roughly 3 million cells into each of three dishes. I then replaced the media in my virus/control cells with 1µg/µL of G418. I split my clone A cells 1:10 and that was it for the day.

6/26
I began making a virus today. I will list the reagents as follows:

Tube Ingredients

A- β4 FSIPPW .41µg/µL= 7.317µL + 9µL Packaging + 1.5mL Opti-Mem media

B- EGFP FSIPPW .904µg/µL= 3.319µL + 9µL Packaging +1.5mL Opti-Mem media

C- 3mL Opti-Mem 72µL Lipofectamine

I waited 5 minutes for the Lipofectamine to incubate, then added 1.5mL of tube C to each other tube, then incubated at room temperature for 20 minutes. Then I replaced the FT media in the plates with 5mL of Opti-Mem media and incubated at 37º for 20 minutes. I then aspirated the media out of the plates and added 3mL of tube A into 1 plate and 3mL of tube B into the other plate and incubated at 37º for 5 hours. Then I added 5mL of FT media (no serum or antibiotics) with 20% FBS to the plates and incubated them at 37º overnight.

Next, I began what is called a Miniprep kit on my bacteria. I spun down 4mL of each tube (15 of them) at 4000 rpm for 5 minutes. I then followed the instructions of the miniprep kit down to the letter, which essentially just isolated the DNA that had been growing inside of the E. Coli. I then digested the Minipreps using the enzymes EcoRI and BamHI and then incubated at 37º for 90 minutes. I ran the digest mix through a 2% agarose gel and picked the three best looking samples after analyzing under a UVP. Sadly, none of the samples yielded the results I was looking for, so I was forced to hope that the DNA I was trying to find was just in too low concentrations to be picked up. I made three 250mL flasks of LB/amp with the samples added (500µL of each) and put them in a high-speed shaker at 37º overnight.

6/27
I made a batch of serum and antibiotic-free FT media, adding 1mM Sodium Pyruvate, 1mM NEAA, and 2mM L-Glutamine. I then replaced the media in the 293 cells with 10mL of 10% FBS DMEM (serum and antibiotic free)

I ran the DNA I had been growing through a Maxiprep kit, which gave me higher concentrations of the DNA. I digested them overnight along with a control FSIPPW using the miniprep reagents.

6/28
I removed the digested lysates from the incubator and ran them on a 2% agarose gel again at 110 volts. I again viewed the gel under a UVP and the results came back negative. This means that I need to redo the experiment all over again.

I split my clone A cells 2:5 and froze down my eGFP cells. I took Friday off and left for wrestling camp on Saturday, not to return until July 5th.