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New Midi Prep kit

We have a new Midi Prep kit. Adam actually got us the Hi -Speed  kit – which should be alot faster.

But, the steps are a little bit different. When you use it – be sure to use the instructions in the box.

HiSpeed Plasmid Midi Kit

http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/hispeedplasmidmidikit.aspx

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.