For anyone that does a great deal of genotyping, the first step is always the isolation and purification of DNA from tissue or tail. I have tried several different kits and buffer systems in an attempt to find the purest, easiest, and least repetitive method of extraction. So when I heard about the USB PrepEase® Genomic DNA Isolation Kit, I thought I would give it a try.
The kit starts with the standard addition of buffer and proteinase K followed by a 55°C incubation. Although the kit claims that 2-4 hours is enough for complete DNA digestion, I have found that at least 6 hours is needed for digestion of hardier tail pieces, and even then there is always some skin and nail tissue left over. Following digestion is the addition of a protein precipitation buffer along with a 5:1 chloroform:isoamyl alcohol solution. This precipitates the protein from the solution, which is left in the lower aqueous phase after vortexing and spinning the sample at high speed for 4 minutes. The upper aqueous phase containing the DNA is collected, isopropanol is added to the solution and the DNA is precipitated out of solution to form a pellet. The pellet is subsequently subjected to an ethanol wash. The DNA pellet must then be dried for about an hour before rehydration with the proprietary DNA precipitation buffer. The yield is quite high, with a typical 30 ug per prep for a tail sample.
This kit works very well and the DNA isolated is pure, as I have never had any problems with downstream PCR reactions. The price per reaction is much less than Qiagen’s DNeasy Kit, which is my normal method of purification and the one I have found gives me the purest DNA. Although this kit gives results without the need for columns, it requires a smelly chloroform addition which requires the need for a fume hood and a lot of clean fresh air. I avoid working with chloroform if I can. That being my number one complaint, I have also never been fond of looking for tiny pellets on the bottom of tubes due to the possibility of a pellet being poured out along with the ethanol washes. There is also the wait time for the pellet to dry before reconstitution buffer can be added and the DNA used. Although the end result is just as good as any other kit, the process involves several steps that I would rather skip. If saving money is a main concern, it is also pretty easy to make your own digestion buffer and there are several protocols that use NaCl or similar compounds, eliminating the chloroform extraction process. This would be even cheaper than buying the USB kit. So, although, the DNA purification result is great, I don’t think I’ll be switching over. But if money is an issue and you’re happy spending a few extra minutes waiting for the pellet to dry and you don’t mind the chloroform odor, this would be a perfect fit.