The UltraClean Microbial DNA Isolation Kit from MoBio Laboratories is a simple to use kit designed to isolate DNA from yeast, fungi, gram negative and gram positive bacteria. It requires approximately 30 minutes to yield high concentration, as well as high quality, DNA. The kit comes with all the necessary reagents including: spin columns, microbeads and collection tubes. The microbeads are the feature that sets this kit apart as they are used to mechanically lyse the cells.
Basically, the protocol states to spin down 1.8 ml of bacterial culture for 30 seconds. I have found that a longer spin is required to adequately pellet the bacteria and assume they used this arbitrary time to decrease the overall time of the procedure. The supernatant is then completely removed and the cells are resuspended in a bead solution along with a lysing reagent. The kit recommends buying the MoBio Vortex adapter to put on a standard vortex. This adapter allows the tubes to remains secure through the 10 minute vortex. I found this adapter worked, but the tubes occasionally flew out and it did not appear to evenly shake every tube. Fortunately, my lab has a mechanical bead beater that holds microcentrifuge tubes and this method works much better and does not require the full 10 minutes. This combination of detergent, mechanical force and heat against the beads leads to lysing with minimal DNA shearing. Then the tubes are centrifuged for 30 seconds to pellet the beads. The supernatant is easily transferred from the beads to a clean tube. Another lysing buffer is added and after 5 minute incubation at 4C, after a brief centrifugation the solid cellular components form a pellet. The supernatant containing the released DNA is bound to a silica spin filter and washed with an ethanol solution. It is imperative to dry the filter completely to remove all ethanol. Finally the DNA is eluted using a certified DNA-free Tris buffer. The kit recommends storing the DNA at -20C as the elution buffer contains no EDTA. The binding capacity of the filter is stated as 20ug per filter.
This kit has become a great addition to my laboratory. I isolate DNA from multi-drug resistant Staphylococcus sp. which are notoriously difficult to lyse because of their thick cell wall. I have used other kits in the past that have taken 6-8 hours to complete and required an additional lysis steps. The mechanical lysing in this kit allows me to completely lyse the bacteria without the need for a 1-2 hour lysostaphin incubation. I tested this by lysing the cells with lysostaphin as usual and performing the protocol as written. Then a second sample was not lysed before going through the protocol. Lysing prior to the stated kit protocol did not increase the yield when using this kit. The simplicity and quickness of this kit make it an invaluable tool for all bacterial labs.
One of the problems I have with this kit, however, is that the manual is very simplistic. It is one page with little or no explanation of the steps. The reagents are not defined and only labeled with numbers. The troubleshooting section is quite basic and the “other UltraClean kits available” section takes up a large portion of the user manual. If DNA shearing becomes a problem the kit does offer an alternative method for lysis. The bead tubes can be heated to 65C for 10 minutes with a 3 second vortex every 3 minutes. This should reduce shearing as well as yield. I have not tried this method and believe it would not be adequate to lyse gram positive organisms.
Overall, the MoBio Laboratories UltraClean Microbial DNA isolation kit is quick, easy and affordable. It has found a method to overcome the lysing issues faced by researchers who work with gram positive and/or resistant organisms. I am pleased with this kit and would highly recommend it to others.
Katrina Williams
PhD student and Lab Specialist, Sr
Division of Infectious Disease
McGuire VA Medical Center