When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.
Procedure
1. TISSUE: Cut ~20 mg tissue (fin or muscle) into small pieces, and place in a 1.5 ml microcentrifuge tube. Add 180 ul Buffer ATL. Grind tissue thoroughly using a disposable plastic pestle. Add 20 ul proteinase K. Mix thoroughly by vortexing, and incubate at 55°C overnight.
2. EMBRYOS: Pool 20 or more embyos in a 1.5mL microcentrifuge tube. Add 180ul Buffer ATL. Grind embryos gently using a disposable plastic pestle.
Add 20 ul proteinase K. Mix thoroughly by vortexing, and incubate at 55°C for 3 or more hours.
After incubation the lysate may appear viscous, but should not be gelatinous as it may clog the DNeasy Mini spin column. If the lysate appears very gelatinous, see the Qiagen “Troubleshooting Guide”, page 47, for recommendations.
After incubation the lysate may appear viscous, but should not be gelatinous as it may clog the DNeasy Mini spin column. If the lysate appears very gelatinous, see the Qiagen “Troubleshooting Guide”, page 47, for recommendations.
3. Add 4 ul RNase A (100 mg/ml), mix by vortexing, and incubate for 2 min at room temperature.
4. Vortex for 15 s. Add 200 ul Buffer AL to the sample, and mix thoroughly by vortexing. Then add 200 ul ethanol (96–100%), and mix again thoroughly by vortexing.
It is essential that the sample, Buffer AL, and ethanol are mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution. Buffer AL and ethanol can be premixed and added together in one step to save time when processing multiple samples.
A white precipitate may form on addition of Buffer AL and ethanol. This precipitate does not interfere with the DNeasy procedure. Some tissues may form a gelatinous lysate after addition of Buffer AL and ethanol. In this case, vigorously shaking or vortexing the preparation is recommended.
A white precipitate may form on addition of Buffer AL and ethanol. This precipitate does not interfere with the DNeasy procedure. Some tissues may form a gelatinous lysate after addition of Buffer AL and ethanol. In this case, vigorously shaking or vortexing the preparation is recommended.
5. Pipette the mixture from step 3 (including any precipitate) into the DNeasy Mini spin column placed in a 2 ml collection tube (provided). Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube.*
6. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500ul Buffer AW1, and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through and collection tube.*
7. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 ul Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube.
It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution. Following the centrifugation step, remove the DNeasy Mini spin column carefully so that the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifugation for 1 min at 20,000 x g (14,000 rpm).
8. Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube (not provided in kit), and pipet 200 ul Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute.
9. Repeat elution once as described in step 7.
* Flow-through contains Buffer AL or Buffer AW1 and is therefore not compatible with bleach. See Qiagen page 8 for safety information.