Myeloid Leukemia

98 Branford and Hughes
and reverse primers (final concentration of 0.3 µM each), 0.375 µL of 5 U/µL
Expand long template enzyme mix, and sterile water to a total volume of 22 µL
(Mix 1). For samples requiring the addition of 2 µL of cDNA (100 ng), prepare a
mix containing the same reagent volumes as above but adjust the volume of water
to 23 µL (Mix 2). The most appropriate positive control to use is a previously
analyzed patient sample of adequate RNA quality with a mid-range BCR-ABL
transcript level (see Note 2).
4. Mix the tubes thoroughly and centrifuge briefly to collect the mix at the bottom
of the tube.
5. Pipet 22 µL of Mix 1 into 0.2-mL tubes for samples requiring 3 µL of cDNA and
23 µL of Mix 2 for samples requiring 2 µL of cDNA into 0.2-mL tubes.
6. In a separate laboratory, thaw the cDNA, mix, and centrifuge briefly. Add 2 µL
or 3 µL of the cDNA to the appropriate 0.2-mL tubes, mix thoroughly, and centrifuge
briefly to collect the mix at the bottom of the tube.
7. Transfer the reactions to the thermal cycler and amplify with the following conditions:
1 cycle at 94°C for 2 min.
10 cycles at 94°C for 10 s, 60°C for 30 s, 68°C for 2 min.
20 cycles at 94°C for 10 s, 60°C for 30 s, 68°C for 2 min—increase the 68°C step
by 20 s at each cycle.
1 cycle at 68°C for 7 min.
8. After completion of the PCR, assess the size of each amplification product by
electrophoresis of 5 µL of the PCR product mixed with 1 µL gel loading buffer
on a 2.0% agarose gel, using the SPP1 size marker. The no-template control
sample should not contain an amplified product. If it does, the PCR should be
repeated. The positive control should have a detectable product, otherwise the
PCR should be repeated (see Note 2). If a product is present of the correct size
(1504 bp for b2a2, 1579 bp for b3a2, and 1641 bp for e1a2), purify the PCR
product using the UltraClean Purification kit according to the manufacturer’s
instructions.
9. If a faint product of the correct size is visible or if no product is present, the
second-step PCR is required. Purify the PCR product using the UltraClean Purification
kit according to the manufacturer’s instructions. Do not purify the notemplate
control.
10. Thaw the PCR reagents and second-step primers (the second-step reverse primer
is the same as the first-step reverse primer) and mix thoroughly before use (except
for the enzyme, which is stored at –20°C until added to the master mix). Centrifuge
briefly to collect the material at the bottom of the tube.
11. Prepare a master mix on ice by adding the following to a 1.5-mL tube for each
patient sample + 1 positive control + 1 no-template control + 1: 0.75 µL MgCl 2,
1.25 µL dNTP (15 mM mix), 2.5 µL 10X Expand long template Buffer 3, 0.15 µL
of the forward and reverse primers (final concentration of 0.3 µM each), 0.375
µL of 5 U/µL Expand long template enzyme mix, and sterile water to a total
volume of 23 µL.
12. Mix the tubes thoroughly and centrifuge briefly to collect the mix at the bottom