Myeloid Leukemia

Chimerism Analysis 283
12. Upon complete (or partial) digestion of the nails, extract the DNA using standard
phenol/chloroform extraction and ethanol precipitation.
3.2.3. DNA Extraction From Flow-Sorted Cell Subsets (see Note 9)
3.2.3.1. VARIANT A: QIAGEN COLUMN EXTRACTION
1. Resuspend with 200 µL PBS, then add 200 µL AL buffer.
2. Vortex for 15 s.
3. Add 20 µL protease, then vortex the solution briefly.
5. Incubate at 70°C for 10 min.
6. Add 210 µL ethanol (absolute), then vortex briefly.
7. Apply the solution to the spin column.
8. Centrifuge and wash the sample according to the standard Qiagen protocol.
9. Collect DNA with 100 µL of elution buffer (see Note 10).
3.2.3.2. VARIANT B: PROTEINASE K LYSIS
1. Collect flow-sorted cells in 50 µL Tris-buffer.
2. Add 10 µL of 100 µg/mL proteinase K (PK).
3. Incubate for a minimum of 1 h at 56°C.
4. Vortex and spin down liquid briefly.
5. Incubate for 10 min at 95°C to inactivate the PK.
6. Vortex and spin down in Eppendorf centrifuge at full speed for 30 s to separate
the DNA supernatant from the cellular debris.
7. Use 10–30 µL of the supernatant as template for the PCR reaction (avoid aspiration
of the cellular debris in the pellet to prevent inhibition of the PCR reaction).
3.3. STR-PCR and Capillary Electrophoresis With Fluorescence-
Assisted Detection for Chimerism Analysis (see Note 11 and Fig. 1)
1. Set up the PCR reactions in a total volume of 50 µL as follows:
dH 2O: 5.5 µµL
10X buffer: 5.0 µµL
MgCl 2 (25 mM): 1.0 µL
dNTPs (from 2.5 mM stocks): 4.0 µL
Primers (from 2.5 pmol/µL stocks): 4.0 µL
Qiagen Taq Polymerase (from 5 units/µL stock): 0.5 µL
DNA (see Note 12): 30.0 µL
Total volume: 50.0 µL
2. Perform PCR amplification under the following cycling conditions:
Initial denaturation at 95°C: 15 min
32 cycles including denaturation at 94°C: 60 s
annealing at 54°C: 45 s
extension at 72°C: 90 s
Final extension step at 72°C: 7 min
Eliminate split peaks by adding an additional extension step at 60°C: 45 min
(see Note 13).