Midi Prep Success

Midi Prep Graph of DNA Concentration

This graph represents the DNA concentration found from the gene that grew in the bacterial culture. This was my second and a half time doing MIDI Prep. The half time, I tossed my supernatant on STEP 7 of the protocol and the first time I completed the MIDI Prep Lab, I got a DNA concentration of .8 ng/ul. Step 4 and 5 went fine during this time of the lab. The solution turned a elegant blue color when the P2 buffer was added to the solution. The second time I completed MIDI Prep, the solution turned a bluish, green on STEP 5 of the lyse to the bacteria cell. This made me a little nervous, because it was suppose to turn an obvious blue color. I talked to the mentors about my solution because I thought it had something to do with my bacteria cells from transformation. My cells were very small and it looked as if they should have been grown up a little longer in the incubator. Despite this, I continued with the lab. After the centrifuge, and resin parts of the lab, I had to spin down my eluted DNA. (Side: In the Resin part of the lab with the MIDI 100 tip, what was happening was a purification of the DNA. After equilibration, the supernatant was poured in the tip and as it flowed the DNA had affinity to the cloth like material close to the filter of the tip. The DNA sat on this material as two washes with QC buffer got all the excess lipids, protein and another cell material that did not get centrifuged out. Next the QF buffer was added to the tip and this took the DNA from the cloth and pulled it into the 15ml conical tube, so that just DNA would be worked with from the steps proceeding elution.)After it was spun down, I looked in the 15 ml tube and saw nothing. From this point, I was nervous. After about ten minutes, I began to panic and almost threw my elute away until a tapeworm looking substance exposed from the bottom of  the tube. It was DNA!!! So, after the isopropanol step, really look at the tube thoroughly because DNA is hard to see and it might be in there. After this I continued on with MIDI Prep and completed the rest on the Lab. Next, it was time to Nanodrop and the first reading was 27.3 ng/ul. I knew this was low, but I asked for clarification anyways. It was low. Now, my mind is really lost because I believed I had a sufficient amount of DNA. After 2 more Nanodrops, I was about to figure out a new plan for MIDI Prep. Then, I asked for clarity from someone as to why my concentration so low.  After this discussion, I vortexed my solution with the Tris-CL Buffer at a pH of 8.5 and centrifuged it so that I could Nanodrop it for the fourth time. On the fourth Nanodrop, I got a DNA concentration of 1280.9 ng/ul. MIDI PREP WORKED!!! This was enough DNA from the bacteria and I could continue on with lab. So, before Nanodropping vortex your solution in the last step for about 10 seconds and centrifuge it for about 30 sec to 1 minute. This DNA obtained in the lab will used for gene insertion into pNIC-bSA4 vector for further analysis in lab.

Labeling plasmid DNA stocks

After midiprep or maxiprep, you want to label your tubes of stock DNA well because you spent a lot of time and effort making it. Also, this DNA will be shared by the whole lab so it should be well labeled.

Label the top and side of tube – use the red sticky dots if necessary (the white squares are lame)

Put:      Initials, Date, type of DNA (pNIC-Bsa4, pGBR22, HSC0041026, etc. ), the concentration in λ ( lambda = micrograms/microliter )    OR  put the concentration  in nanograms/microliter. If it contains a gene inside of a vector – you should put the Gene name and the Vector name (e.g.     DHFR in pNIC-Bsa4)

Resituation and Starting Midiprep

Happy first week back everyone! This week wasn't too eventful but
involved some vital planning for the next few weeks for me. Upon
returning to UT, I was surprised to hear that class was canceled
because Dr.B's son was on the way(congratulations!). But,
nonetheless, the research had to go on. On Wednesday,I added
glycerol to my pNIC-Bsa4 + CA7 protein for long term storage and
double checked its concentration - 1.36 mg/ml (29.147 uM, calculated
using Beer's Law). Additionally, I verified the presence of the
master plate from which I originally got my protein and ensured
correct labeling and storage. On Wednesday, I also deliberated
which direction to go in - repeat my enzyme assay, grow up more
protein, or Midiprep the protein I already had. Initially, I planned
to grow up more protein but, realizing the difficulty in stopping
over the Labor Day weekend, I talked to super mentor Adam and
decided to Midiprep instead. On Thursday,I began the Midiprep and
started the overnight culture of my pNIC-Bsa4 with CA7. On Friday,
I decanted and spun down my overnight culture and stored it in the
-20C fridge over the weekend. Also, I organized my notebook and
made sure everything was up to date. I plan to continue my Midiprep
next week, alongside performing the refreshers Dr. B has made and
beginning my virtual screening (potentially).