The mini-prep procedure extracts plasmid DNA (small rings of double stranded DNA) from bacteria. We use the bacteria for making many copies of the plasmid of interest. Basically, we feed it and in return get amplification of small amount of plasmids into large quantities we can work with.
While running a mini-prep is day-to-day routine in many labs, doing it the first required to make sure we have all the ingredients. It was also a first time for me in teaching a protocol. So Avital had to see me learn how to teach...
Ayelet and Avital grew the bacteria overnight from stock we had in our refrigerator. This morning Avital and I started by centrifuging the bacteria into small pellets, and then remove the media by pipetting.
(photos courtesy of Ruty)We then started with the kit instructions for the protocol. As you can see, the little card summarizes the steps.
The heart of the kits are these mini filter tubes that can bind DNA under certain conditions and release it when washed with a specific buffer. This means that during the first washing stages we run the supernatant from the cells and the DNA binds to the filter while all other molecules pass through, and then in the last wash the DNA is released into the small sample tube.
To carry these steps we use a small centrifuge. The centrifugal force serves to drive all liquid from the top chamber to the tube below, moving it through the filter.
Once we finished the mini-prep we wanted to ensure that it worked. First, off as a test of the plasmid carrying bacteria that we were using, and second to teach ourselves the careful steps in checking our procedure.
We did two test. The first is to run the samples through a spectrophotometer. Unlike the use of spectrophotometer for measuring cell density, here we use it to check that we get absorption at the right wavelength (260nm) which indicate that we have DNA. Since the final sample is very small (50 microliters), we used a cool device called NanoDrop that is a spectro-photometer on a drop of 1 microliter. See the liquid at the tip of the pipette here:
The NanoDrop showed that all of our samples had very nice spectra, suggesting no contamination. It also computed for us the concentration of DNA in the samples.
Next, we wanted to make sure we didn't get any genomic DNA or other stuff. For this, we run a gel electrophorsis. We opened the gel device from its wraps, prepared TAE buffer, and "cooked" agarose gel in our lab microwave. We then poured into the mold, using a "comb" to create small wells in the gel.
After the gel solidified, we filled the device with buffer. To load the gel, we needed to combine 1 microliter of sample with a dye that will highlight the DNA. Ayelet taught us a neat trick to prepare the mix using small droplets on a parafilm.
We then used a pipette to load each of these small drops into one of the "wells" created by the comb in the gel mold. This meant aiming the tip into a small rectangle in a translucent gel within a water bath.
Once the gel is loaded, we connected the bath to electrodes, and run 120 volts through it. The bubbles from the electrodes show that proper electric field is established.
To our delight, the gels result showed nice clear bands in our samples.