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Animal tail DNA extraction for real time PCR genotyping

For some experiments, we may want to cross hemizygous transgenic mice to obtain homozygous animals which contain the transgene in both alleles. I usually use Real-time PCR to screen the homozygous vs hemizygous transgenics. For this, I will need cleaner tail DNA while the preparation from Viagen solution does not work for this application.

Reagents

• DNeasy Blood & Tissu kit from Qiagen #69506.

1. Cut tails (0.2-0.5 cm) from animals.

2. Prepare tail lysis buffer by adding proteinase K solution (within the kit) to ATL solution. For each tail, I use 200 ul lysis buffer which contains 20 ul Proteinase K solution (10 X) in 180 ul ATL solution.

3. Incubate at 55 C overnight. I usually put all the tubes into the hybridization glass bottle and keep rotating in the Isotemp incubator.

4. The next morning, vortex the tubes to mix the solution. Add 400 ul solution which contains 200 ul AL solution and 200 ul 100% ethanol. Vortex vigorously.

5. Pour the entire tube into a column. There will be some leftover in the tube. You do not need transfer all of it since the yield is quite well. Make sure to label the column accordingly.

6. Spin down 14K RPM for 1 min at RT.

7. Discard the flowthrough and add 500 ul AW1. Spin down at 14K RPM for 1 min at RT. Note: Add ethanol to AW1 concentrate before using it.

8. Discard the flowthrough and add 500 ul AW2. Spin down at 14K RPM for 1 min at RT. Note: Add ethanol to AW2 concentrate before using it.

9. Discard the flowthrough. Spin down at 14K RPM for 1 min at RT to remove all carryover buffers.

10. Transfer the column to a new eppendorf tube and add 200 ul buffer AE to the center of the column. Sit on bench for 1 min.

11. Spin down 14K RPM for 1 min at RT to collect the DNA.

12. This DNA solution can be stored at RT for quite a while. I have tested it after 2 weeks which does not affect the quality for real time PCR.