NOTE: The QIAGEN protocol for suggests that you use a mortar and pestle to crush part of the insect into a powder, but I have found that this is unnecessary.
- Remove a piece of leg that is about 1cm long and put it into a 1.5 or 1.7ml microcentrifuge tube. Make sure to follow Flame Sterilization or the Eliminase for Sterilization between samples.
- Add 180ul ATL Buffer to the sample.
- Add 20ul proteinase K and vortex the sample until mixed.
- Incubate the sample in the shaking incubator at 56°C for overnight.
- Vortex the sample for 15 sec. Add 200ul Buffer AL and vortex it (a white precipitate will most likely form) and add 200ul ethanol (96-100%) and vortex until it is homogeneous (there should be a lot less white precipitate).
- Pipette the mixture (set the pipette to 600ul) into a spin column and make sure to label the cap, and centrifuge it at 8000 rpm for 1 min (you have to set the centrifuge to 2 min, and once it reaches 8000 rpm start your watch and time it for 1 min).
- Throw out the collection tube full of liquid and put the spin column into a new tube. Add Buffer 500ul AW1 and centrifuge at 8000 rpm for 1 min.
- Throw out the collection tube full of liquid and put the spin column into a new tube. Add Buffer 500ul AW2 and centrifuge at 130 000 rpm for 5 min.
- Throw out the collection tube full of liquid and remove the spin column carefully as not to let it touch the liquid and put the spin column into a 1.5 or 1.7 ml microcentrifuge tube (not from the kit, one with a cap) and label it.
- Add 100ul Buffer AE (elution buffer) and let it sit at room temperature for 1 min. Centrifuge at 8000 rpm for 1 min.
- The liquid in the bottom of the microcentrifuge tube is your DNA.
Return to Manual Extraction.
The next step is the PCR Procedure.
Updated May 3 2009