Anthony and I are continuing the extraction of the pBR322 that we started last Wednesday. We completed the procedure up though collecting it on a gel. A picture of the final gel can be found here. Today we are extracting the DNA from the gel. To do this we use a kit and the general procedure is:
- Weigh the mass of the gel sample We need to know the weight of the sample so that we know how much of the QG buffer to add to dissolve the agarose. To do this, we just weigh out one of the sample tubes, then place the agarose sample inside of it and measure out the difference in the weight.
- Add the Buffer QC We add 300 μL of the buffer for every 100 μg of the agarose. The buffer serves to dissolve the agarose away. We warm the tube at 50°C and vortex it every couple of minutes to help dissolve the agarose. This usually takes about ten minutes.
- Spin the Sample Down You add up to 800μL at a time of the solution to the specially designed collection tube. You then spin the sample down at 13,000 rpm for one minute, collecting the DNA in the little frit, and discard the excess solution in the bottom of the tube.
- Add Buffer QC Add 0.5 mL of Buffer QC to the column and centrifuge it for a minute, discarding the waste.
- Wash with Buffer PE Add 0.75 mL of Buffer PE to the column and spin it for a minute, also discarding the waste.
- Extra Spin Down We then spin the collection column down for an additional minute, without any added reagents. This collects the waste which can then be discarded. The top part of the collection column is then placed into a clean 1.5 mL centrifuge tube that can collect the DNA.
- Elute the DNA Add 50 μL of Buffer EB to the column, directly onto the frit without touching it with the pipette tip. Let the tube stand for a minute, then centrifuge it for another minute.
- Determine Concentration Use the Nanodrop to determine both the concentration and volume of DNA in the container.